Short peptides useful for treatment of ischemia/reperfusion injury and other tissue damage conditions associated with nitric oxide and its reactive species

ABSTRACT

This invention discloses isolated short peptides comprising the amino acid sequence Cys-Glu-Phe-His (CEFH) and analogs thereof as well as compositions comprising CEFH peptides and analogs thereof. The CEFH peptides disclosed herein are effective in mediating the denitration of 3-nitrotyrosines (3-NT) in cellular proteins thereby preventing tissue damage associated with excess nitric oxide (NO) and its reactive species. The CEFH peptides disclosed herein are useful in the treatment of ischemia/reperfusion (I/R) injury of various tissues (e.g., I/R injury of heart muscle associated with heart attack or cardiac surgery, I/R injury of brain tissue associated with stroke, I/R injury of liver tissue, skeletal muscles, etc.), septic shock, anaphylactic shock, neurodegenerative diseases (e.g., Alzheimer&#39;s and Parkinson&#39;s diseases), neuronal injury, atherosclerosis, diabetes, multiple sclerosis, autoimmune uveitis, pulmonary fibrosis, oobliterative bronchiolitis, bronchopulmonary dysplasia (BPD), amyotrophic lateral sclerosis (ALS), sepsis, inflammatory bowel disease, arthritis, allograft rejection, autoimmune myocarditis, myocardial inflammation, pulmonary granulomatous inflammation, influenza- or HSV-induced pneumonia, chronic cerebral vasospasm, allergic encephalomyelitis, central nervous system (CNS) inflammation,  Heliobacterium pylori  gastritis, necrotizing entrerocolitis, celliac disease, peritonitis, early prosthesis failure, inclusion body myositis, preeclamptic pregnancies, skin lesions with anaphylactoid purpura, nephrosclerosis, ileitis, leishmaniasis, cancer, and related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119, to U.S.Provisional Application Ser. No. 60/887,314 filed Jan. 30, 2007, thedisclosure of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Research and development leading to certain aspects of the presentinvention were supported, in part, by a grant from NIH AI60762.Accordingly, the U.S. government may have certain rights in theinvention.

TECHNICAL FIELD OF THE INVENTION

This invention relates to isolated short peptides comprising the aminoacid sequence Cys-Glu-Phe-His (CEFH; SEQ ID NO: 1) and analogs thereofas well as compositions comprising CEFH peptides and analogs thereof.The CEFH peptides disclosed herein are effective in mediating thedenitration of 3-nitrotyrosines (3-NT) in cellular proteins therebypreventing tissue damage associated with excess nitric oxide (NO) andits reactive species. Thus, CEFH peptides disclosed herein are usefulfor treatment of diseases associated with excess NO and its reactivespecies, which diseases include, but are not limited to, acute andchronic disorders such as ischemia/reperfusion (I/R) injury of varioustissues (e.g., I/R injury of heart muscle associated with heart attackor cardiac surgery, I/R injury of brain tissue associated with stroke,I/R injury of liver tissue, skeletal muscles, etc.), septic shock,anaphylactic shock, neurodegenerative diseases (e.g., Alzheimer's andParkinson's diseases), neuronal injury, atherosclerosis, diabetes,multiple sclerosis, autoimmune uveitis, pulmonary fibrosis,oobliterative bronchiolitis, bronchopulmonary dysplasia (BPD),amyotrophic lateral sclerosis (ALS), sepsis, inflammatory bowel disease,arthritis, allograft rejection, autoimmune myocarditis, myocardialinflammation, pulmonary granulomatous inflammation, influenza- orHSV-induced pneumonia, chronic cerebral vasospasm, allergicencephalomyelitis, central nervous system (CNS) inflammation,Heliobacterium pylon gastritis, necrotizing entrerocolitis, celliacdisease, peritonitis, early prosthesis failure, inclusion body myositis,preeclamptic pregnancies, skin lesions with anaphylactoid purpura,nephrosclerosis, ileitis, leishmaniasis, cancer, and related disorders.

BACKGROUND OF THE INVENTION

Myocardial infarction is one of the most widespread and serious healthproblems in Western society. In 2002, in the United States alone, overone million individuals suffered a myocardial infarction with over 25%fatality. During a heart attack, one or more of the arteries that supplythe heart becomes blocked by a blood clot, usually at the site of fattydeposits known as arteriosclerosis. When victims are rushed to ahospital, blood flow is restored (the process known as reperfusion)either by drugs that dissolve clots or by angioplasty. Tissue salvage,however, is severely limited by free radicals and inflammatoryresponses, which cause as much as 80% of the damage during reperfusion(ischemia-reperfusion [I/R] injury). This, in turn, raises the risk oflethality and long-term complications.

A primary outcome of damage resulting from I/R injury is chroniccongestive heart failure. Over 22% of male and 46% of female myocardialinfarction victims will be disabled with congestive heart failure withinsix years following their heart attack. As the average age of thepopulation increases and as survival following myocardial infarctionimproves, congestive heart failure will grow in importance. Followingdiagnosis of congestive heart failure, prognosis is poor. 12% ofpatients die within three months of diagnosis, 33% die within one year,and 60% die within five years.

Apart from accidental heart attacks, I/R injury is also a common outcomein cardiac surgery, leading to a spectrum of damage includingarrhythmias, post-ischemic myocardial dysfunction, and cardiogenicshock. Furthermore, I/R injury is not limited to heart muscle, and alsofrequently occurs in the brain (stroke), liver, skeletal muscles andother organs.

Although the mechanism of I/R injury is not fully understood, a largebody of evidence implicates a dual role for nitric oxide (NO) in thisprocess. While NO is a potent cardioprotector, its excessiveaccumulation in ischemic tissue leads to the formation of reactivenitrogen species (e.g., peroxynitrite) that promote tissue injury bynitrating proteins. NO is a signaling molecule that is involved in amultitude of physiological processes including neurotransmission, immuneregulation, vascular smooth muscle relaxation, and inhibition ofplatelet aggregation. See, e.g., Moncada S., Ann. N.Y. Acad. Sci. 1997;811: 60-67; Ischiropoulos H., Arch Biochem Biophys 356: 1-11, 1998;Stamler et al., Cell 2001; 106: 675-683; Bian et al., J Pharmacol Sci.2006; 101:271-9.

Depending upon the rate, timing, and spatial distribution of NOproduction, as well as the chemical microenvironment (e.g., presence ofreactive oxygen species and redox status of the cell), NO acts either asa direct signaling messenger or as an indirect toxic effector via theformation of various reactive nitrogen species such as, e.g.,peroxynitrite anion (ONOO⁻) and nitrogen dioxide (.NO₂), formed assecondary products of .NO metabolism in the presence of oxidantsincluding superoxide radicals (O₂.⁻), hydrogen peroxide (H₂O₂), andtransition metal centers. See Radi, Proc. Natl. Acad. Sci. USA, 2004,101(12): 4003-4008.

NO is synthesized enzymatically from L-arginine by the enzymenitric-oxide synthase (NOS) in almost all tissues of the body, includingbrain, peripheral nervous system, smooth muscle, kidney, vascular, lung,and uterus.

A large body of evidence has established the role of NO in thepathogenesis of inflammatory, infectious, and neurodegenerativediseases. The detrimental role of NO is rooted in the ability of itsreactive metabolites to alter the function of biological macromoleculesvia covalent modifications of protein tyrosine, cysteine and tryptophaneamino acid residues.

Cysteines and tryptophanes can be nitrosated to form S—NO and N—NO,respectively. These nitrosoderivatives are readily reversible (and formSH and NH₂) in the presence of free thiols.

In contrast, tyrosine nitration has been considered to be anirreversible modification in vivo. Tyrosine nitration is mediated byreactive nitrogen species such as peroxynitrite anion (ONOO⁻) andnitrogen dioxide (.NO₂). Once nitrated at tyrosine, proteins are thusthought to be irreparably damaged. Tyrosine nitration may affect proteinstructure and lead to loss of protein function or to a constitutivelyactive proteins. For example, nitration of a tyrosine residue mayprevent the subsequent phosphorylation of that residue. Alternatively,nitration of tyrosine residues may stimulate phosphorylation and resultin constitutively active proteins. Furthermore, tyrosine nitration maychange the rate of proteolytic degradation of nitrated proteins andfavor either their faster clearance or accumulation in cells. See, e.g.,Turko and Murad, Pharmacol. Reviews, 2002, 54(4): 619-634.

3-nitrotyrosine (3-NT) in body fluids and tissues has served as abiomarker of the involvement of NO in acute and chronic disorders suchas I/R injury, atherosclerosis, diabetes, septic shock, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, pulmonary fibrosis,amyotrophic lateral sclerosis (ALS), inflammatory bowel disease,arthritis, allograft rejection, autoimmune myocarditis, pulmonarygranulomatous inflammation, and cancer. Reviewed in, e.g.,Ischiropoulos, Arch. Biochem. Biophys., 1998, 356(1): 1-11; Turko andMurad, Pharmacol. Reviews, 2002, 54(4): 619-634; Radi, Proc. Natl. Acad.Sci. USA, 2004, 101(12): 4003-4008.

As previously observed by the present inventors and co-workers (seeRafikova et al., [Abstract] Nitric Oxide: Biology & Chemistry,2006,14:A67), tissue NO level is critical in the fate of cardiac tissueduring I/R injury. Rats subjected to 30 minutes of myocardial ischemia(MI) and treated with sodium nitrite (5 mg/kg i.v.), infused 1 minuteprior to ischemia set up, showed a significant expansion of I/R infarctsize and myocardial tissue 3-NT accumulation compared with salinetreated controls. In contrast, the use of NOS inhibitor L-NAME (50 mg/kgi.p.) provided the reduction in infarct size and 3-NT. It was alsodemonstrated that lowering tissue level of NO beyond certain point mayalso result in an enhanced myocardial damage. Thus, there exists anoptimal tissue NO content that provides a minimal cell injury. Larger orsmaller amounts of tissue NO are progressively more harmful probably dueto either initiation of nitrosative stress or lack of NO antioxidantactivity. For example, an excess of NO can lead to the formation ofreactive nitrogen species, protein nitration, endothelial dysfunction,PARP and MMP activation, and mitochondrial respiration inhibition. Theseeffects can lead to flow occlusion, apoptosis, necrosis, and inhibitionof contractile function. However, a deficit of NO can also lead todetrimental effects since NO is a potent cardioprotector as it, e.g.,induces cGMP synthesis, inhibits cytokine expression, and serves as ageneral antioxidant by intercepting oxygen radicals. As a result, NO inmoderate amounts can improve perfusion, inhibit platelet aggregation,inhibit apoptosis, and increase ischemic tolerance.

The majority of current therapies for I/R injury, such as antiplateletagents, anticoagulants, clot-dissolving drugs, vasodilators, and PTCA,target the occluded coronary artery rather than the ischemic tissue perse. Beta-blockers, which act by decreasing a tissue's O₂ demand, are theonly commercially available drugs that are protective against I/Rdamage.

Thus, there remains an unmet need in the art for safe and effectivedrugs that reduce the tissue damage associated with ischemia/reperfusion(I/R) injury of various tissues as well other types of tissue damageassociated with septic shock and neurodegenerative diseases.

SUMMARY OF THE INVENTION

The present invention fulfills these and other related needs byproviding isolated short peptides comprising the amino acid sequenceCys-Glu-Phe-His (CEFH; SEQ ID NO: 1) as well as analogs and derivativesthereof, which peptides efficiently denitrate cellular proteins and thusprevent tissue damage associated with excess nitric oxide (NO) and itsreactive species. The peptides of the invention are characterized by aunique combination of stacking, ionic and hydrophobic interactions thatallow them to efficiently transfer the NO₂ group from the proteintyrosine to its thiol group and in this way offer an unprecedented levelof protection against ischemia/reperfusion (I/R) injury and otherconditions associated with excess NO and its reactive species.

The peptides of the present invention are preferably short to allow highaccessibility to various parts of a target protein. More preferably,such peptides are less than eight (8) amino acids long, most preferably,such peptides are four (4) amino acids long. Due to their small size,the peptides of the invention can be easily delivered to essentially alltissues and cells of the body, including cells and tissues separated bythe blood-brain barrier (BBB).

In a specific embodiment, the peptide of the invention has the sequenceCys-Glu-Phe-His (L-CEFH peptide; SEQ ID NO: 1). In another embodiment,the peptide of the invention has the sequence Cys-Glu-Phe-His (SEQ IDNO: 1) and consists of only D-amino acids (D-CEFH peptide). In yetanother embodiment, the peptide of the invention has the sequenceCys-Glu-His-His (CEHH peptide; SEQ ID NO: 2). In further embodiments,the peptide has the sequence Cys-Glu-Phe-His-Cys-Glu-Phe-His (CEFH×2peptide; SEQ ID NO: 3) or contains one or more CEFH peptides fused toone or more CEHH peptides, one or more CEFH peptides fused together, orone or more CEHH peptides fused together. One skilled in the art canenvision additional permutations and combinations of the CEFH and CEHHpeptides that are within the scope of the present invention, includingboth linear and cyclic peptides.

In a second embodiment, the invention provides a compound of formula I,

Formula I

or a pharmaceutically acceptable salt thereof, wherein the dotted lineis a bond or absent; R¹, R², R³ are independently —C(O)NH—, -(AA)_(n)-and —C(O)-(LL)_(n)-NH—; when the dotted line is a bond, R^(4a) andR^(4b) together are —C(O)NH—, -(AA)_(n)- or —C(O)-(LL)_(n)-NH—; when thedotted line is absent, R^(4a) and R^(4b) are not the same and areindependently selected from —C(O)OH and —NH₂; AA is a natural orunnatural amino acid, LL is a linker selected from —(CH₂)_(m)—,—(CH₂CH₂O)_(m)—, —(CH₂CH₂S)_(m)— and —(CH₂CH₂NH)_(m)—, B¹, B², B³ and B⁴are not the same and are independently selected from

R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from H, C₁-C₆ alkyl,halogen, hydroxy, cyano, nitro, nitoso, amino, sulfhydryl, C₁-C₆ alkoxy,—C(O)O—C₁-C₆ alkyl, —C(O)O—C₁-C₆ aryl, substituted or unsubstitutedaryl, substituted or unsubstituted 5 to 7-membered heterocyclic ring,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted arylcarbonyl, substituted orunsubstituted alkylcarbonyl and substituted or unsubstitutedaminocarbonylalkyl; each occurrence of n is independently an integerfrom 0 to 5; each occurrence of m is independently an integer from 1 to25; and each occurrence of p is independently an integer from 0 to 6.

In another embodiment, In another embodiment, B¹, B², B³ and B⁴ areselected from

In a further embodiment, B¹, B², B³ and B⁴ are selected from histidine,phenylalanine, glutamic acid and cysteine

In one embodiment R¹, R², R³ and R⁴ are all peptide bonds and thecompound is the cyclic tetrapeptide CEFH.

In another embodiment, one of R¹, R², R³ or R⁴ is —NH₂ and —COOHcomprising the N— and C-terminus groups of a linear compound. Forexample, in the case where R¹ is —NH₂ and —COOH, the compound is lineartetrapeptide H₂N—CEFH—COOH. In the case where R² is —NH₂ and —COOH, thecompound is linear tetrapeptide H₂N—HCEF—COOH. In the case where R³ is—NH₂ and —COOH, the compound is linear tetrapeptide H₂N—FHCE-COOH. Inthe case where R⁴ is —NH₂ and —COOH, the compound is linear tetrapeptideH₂N-EFHC—COOH.

In yet another embodiment, R¹, R², R³ or R⁴ are one or more amino acids.For example, if R² is alanine the compound is cyclic-CEFAH. If R² isarginine and R³ is alanine the compound is cyclic-CEAFRH.

In a further embodiment, one or more R¹, R², R³ and R⁴ can be of adifferent chemical nature then a peptide bond or an amino acid. Forexample, the compound may contain an alkyl bridge —(CH₂)_(n)—,polyetheylene glycol —(CH₂CH₂O)_(m)—, —(CH₂CH₂S)_(m)— or—(CH₂CH₂NH)_(m)—, as well as many other derivatives. For example,linkers R¹, R², R³ or R⁴ may be conjugated with additional molecules inorder to achieve a medicinal or pharmacological goal, such as drugtargeting and delivery, increasing in vivo lifespan or improved removalfrom tissues and organs.

In another embodiment, residues R⁵, R⁶, R⁷, R⁸ and R⁹ are modified inone or more available positions with functional group that areindependently selected from H, C₁-C₆ alkyl, halogen, hydroxy, cyano,nitro, nitoso, amino, sulfhydryl, C₁-C₆ alkoxy, —C(O)O—C₁-C₆ alkyl,—C(O)O—C₁-C₆ aryl, substituted or unsubstituted aryl, substituted orunsubstituted 5 to 7-membered heterocyclic ring, substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl,substituted or unsubstituted arylcarbonyl, substituted or unsubstitutedalkylcarbonyl and substituted or unsubstituted aminocarbonylalkyl, whichmay increase denitration activity of the whole molecule.

In a specific embodiment, the amino acid cysteine is replaced withhomo-cysteine, histidine is replaced with arginine, phenylalanine isreplaced with tyrosine, and glutamic acid is replaced with asparticacid. Further, any of these amino acids may be substituted withadditional chemical groups.

In another embodiment, the relative positions of the amino acid residuesmay be altered. For example, compounds such as cyclic-CEFH, cyclic-CEHF,cyclic-CFEH, cyclic-CFHE, cyclic-CHFE and cyclic-CHEF are envisioned.

AA is a natural or unnatural amino acid. In one embodiment, the aminoacid is attached via a peptide bond.

The peptides of the present invention can be used at concentrations thatare both therapeutically effective and pharmaceutically acceptable. TheL-CEFH peptide of the present invention is preferably used to treat orprevent tissue damage in vivo at 0.1-3.5 mg/kg, most preferably at 0.7mg/kg. The D-CEFH peptide of the present invention is preferably used at0.01-0.5 mg/kg, most preferably at 0.1 mg/kg.

Still further aspects of the present invention provide methods forgenerating and/or identifying novel peptides having the same or betterfunctional characteristics than the CEFH peptides of the invention usingthe following methods aimed at preserving or improving the uniquecombination of stacking and/or ionic and/or hydrophobic interactionsthat allow the CEFH peptides to efficiently mediate the protein tyrosinedenitration:

-   (1) combinatorial shuffling of the relative positions of CEFH amino    acids;-   (2) addition of a linker (e.g., homocysteine) instead of cysteine    (*CEFH) to provide additional spatial flexibility and increase the    reach of SH group;-   (3) introduction of electropositive substitutions (e.g., CH₃, C₂H₅,    tret-Butyl, etc.) into the benzene ring of phenylalanine (CEF*H);-   (4) introduction of tryptophan or cyclic aromatic groups (both    natural and synthetic, e.g., naphtalene, tyrosine, and histidine)    instead of phenylalanine (CE[F−>AR*]H);-   (5) substitution of phenylalanine to histidine with or without an    electropositive substitution (e.g., CH₃, C₂H₅, tret-Butyl, etc.)    (CE[F−>H]H or (CE[F−>H*]H);-   (6) modification that increases pKa of glutamic acid (e.g., OH, NO₂,    or halide in alpha-position) (CE*FH);-   (7) substitution of glutamic acid with aspartic acid with or without    modifications that increase its pKa (e.g., OH, NO₂, or halide in    alpha-position) (C[E−>D]FH or C[E−>D*]FH).

The present invention further provides in vitro and in vivo methods forfunctional testing of the novel denitrating peptides generated using theabove methods, comprising:

-   (1) in vitro testing by adding the peptide to a nitrated protein    having 3-NT and monitoring the disappearance of 3-NT;-   (2) testing by adding the peptide to a cell culture treated with NO    and/or reactive NO species (e.g., subject to NO/ONOO-exposure) and    measuring cell survival by methods such as, e.g., an MTS-based assay    (using the MTS reagent available from Promega, Madison, Wis.) or the    direct counting of apoptotic cells using flow cytometry;-   (3) testing by adding the peptide to a cell culture treated with NO    and/or reactive NO species (e.g., subject to NO/ONOO-exposure) and    measuring the disappearance of 3-NT;-   (4) in vivo testing by administering the peptide to an animal model    of a relevant disease (e.g., I/R injury, septic shock, Alzheimer's    disease, etc.) and monitoring the extent of tissue damage and    disease progression;-   (5) in vivo testing by administering the peptide to an animal model    for I/R injury and determining the size of the infarct using a    p-nitro-blue tetrazolium (NBT)-based assay while also monitoring    functional parameters such as heart rate, mean arterial blood    pressure, cardiac output, etc.

Useful compounds of the present invention are not limited to peptidesincorporating natural and/or non-natural amino acids. A number ofnon-peptide molecules having similar functional properties can bedeveloped to incorporate disparate chemical functional groups within asingle molecule. Chemical functionality comprising these molecules aswell as peptides of the invention would include (i) at least one thiolgroup (for example, cysteine or homocysteine), (ii) at least one polargroup (for example, a functional group with a measurable dipole moment,including, but not limited to, carbonyl groups such as in ketones,esters, or amides, imine groups alone or in heterocycles, cyano groups,guanidine groups, amidine groups, etc. as in serine, threonine, lysine,arginine, histidine, tyrosine, tryptophan, glutamic acid, aspartic acid,glutamine or asparagine, cysteine or methionine), (iii) at least oneproton donor (such as an alcohol, carboxylic acid, hydroxyl amine,heterocyclic or heteroaromatic NH or OH as in serine, threonine, lysine,arginine, histidine, tyrosine, tryptophan, glutamic acid, aspartic acid,glutamine or asparagine), and (iv) at least one aromatic group (forexample, carbocyclic or heteroaromatic groups as in tyrosine,tryptophan, histidine or phenylalanine). It is envisioned that thesechemical groups may be combined into a single functional group (forexample tyrosine, tryptophan, histidine, glutamic acid, aspartic acid,glutamine, asparagine, arginine, and lysine) or be comprised indifferent portions of the molecule.

The present invention also provides compositions comprising one or morecompounds of the invention and a pharmaceutically acceptable carrier,excipient, and/or diluent. Such compositions may further compriseadditional active ingredients having a cumulative effect such as smallthiols (e.g., lipoic acid, homocysteine, N-acetylcysteine [NAC],thioredoxin [TRX], and Bucillamine).

In conjunction with these compounds and compositions, the presentinvention provides methods for use of such compounds and compositions to(i) stimulate denitration of 3-nitrotyrosine in a cell or tissue of ananimal, (ii) prevent cell death induced by nitric oxide and its reactivespecies, (iii) prevent ischemia-reperfusion injury in a tissue of ananimal, and/or (iv) treat a disorder manifested by accumulation of3-nitrotyrosine in an animal, including, but not limited to, tissuedamage associated with I/R injury of various tissues (e.g., I/R injuryof heart muscle associated with heart attack or cardiac surgery, I/Rinjury of brain tissue associated with stroke, I/R injury of livertissue, skeletal muscles, etc.), septic shock, anaphylactic shock,neurodegenerative diseases (e.g., Alzheimer's and Parkinson's diseases),neuronal injury, atherosclerosis, diabetes, multiple sclerosis,autoimmune uveitis, pulmonary fibrosis, oobliterative bronchiolitis,bronchopulmonary dysplasia (BPD), amyotrophic lateral sclerosis (ALS),sepsis, inflammatory bowel disease, arthritis, allograft rejection,autoimmune myocarditis, myocardial inflammation, pulmonary granulomatousinflammation, influenza- or HSV-induced pneumonia, chronic cerebralvasospasm, allergic encephalomyelitis, central nervous system (CNS)inflammation, Heliobacterium pylori gastritis, necrotizingentrerocolitis, celliac disease, peritonitis, early prosthesis failure,inclusion body myositis, preeclamptic pregnancies, skin lesions withanaphylactoid purpura, nephrosclerosis, ileitis, leishmaniasis, cancer,and related disorders. In related aspects, the present inventionprovides isolated polynucleotides that encode the peptides of thepresent invention as well as recombinant vectors and host cells (botheukaryotic and prokaryotic) that have been genetically modified toexpress or overexpress the peptides of the present invention.

In a separate embodiment, the present invention provides a method fortreating ischemia/reperfusion (I/R) injury and treating/reducing invitro and in vivo other types of tissue damage associated with thediseases mentioned above by exposing nitrated proteins to excess ofsmall thiols. Examples of small thiols useful in the methods of theinvention include, without limitation, homocysteine, N-acetylcysteine[NAC], lipoic acid, thioredoxin [TRX], and Bucillamine. The presentinvention also provides combination treatments using peptides of theinvention and small thiols.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All publications, patents, and patent applications citedherein, whether supra or infra, are hereby incorporated by reference intheir entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows interaction of the L-CEFH peptide withprotein 3-NT. The chemical structure of the peptide is shown in bold.The scheme shows how a unique combination of stacking, ionic andhydrophobic interactions allow the peptides of the present invention toefficiently transfer the NO₂ group from the protein tyrosine to itsthiol group.

FIG. 2 is a dot-immunoblotting of a reaction mixture using antibodiesagainst 3-NT showing the time course of L-CEFH peptide-mediateddenitration of peroxynitrite (ONOO)-nitrated albumin. Essentiallycomplete denitration (>90%) was achieved after 10 minutes of treatmentwith 20 μM L-CEFH peptide solution containing 10 mM Tris HCl pH 7.2 at atemperature of 25° C.

FIG. 3 is a bar graph showing that, as compared to “no treatment”control, both cysteine (Cys, at 5mM) and L-CEFH peptide (4P, at 20 μM)improve HeLa cell survival (by 60-80%) upon NO/ONOO-exposure, when addedto HeLa cell culture for 40 minutes as measured by an MTS-based assay(available from Promega, Madison, Wis.). The L-CEFH peptide showsefficacy equivalent to cysteine, when used at 250 times lowerconcentration than cysteine.

FIG. 4 is a bar graph showing that L-CEFH peptide prevents myocardialischemia-reperfusion (MI/R) injury in a rat model of myocardialinfarction. Tested rats received either vehicle (control, n=6) or L-CEFHpeptide (4P, 0.7 mg/kg; n=9) i.v. 5 minutes after beginning ofreperfusion. Infarct size (necrotic area NA) is expressed as apercentage of the area at risk (AR). The decrease in the infarct size inthe presence of the L-CEFH peptide is 3-5 fold as compared to thecontrol.

FIG. 5 is a bar graph showing that L-CEFH peptide prevents accumulationof 3-NT in a rat model of MI. Hearts from the experiment in FIG. 4 wereanalyzed for the presence of 3-NT by dot-immunoblotting of the totalprotein from the heart tissue corresponding to the area at risk withanti-3-NT antibodies (see images under the graph). The vertical graphaxis shows the percentage (%) of nitrated protein in control animals (noI/R) and peptide-treated I/R animals in relation to the non-treated I/Ranimals. As compared to the control, in the peptide-treated I/R animals,the 3-NT accumulation is reduced by about 70%.

FIG. 6 is a bar graph showing that D-CEFH peptide has a superioractivity in preventing MI/R injury as compared to L-CEFH peptide in arat model of MI. Under the same experimental conditions as in FIG. 4,different concentrations of D-CEFH peptide (0, 0.02, 0.05, 0.1, 0.14,0.35, and 0.7 mg/kg; n<4 for each concentration) were administered i.v.Infarct size (necrotic area NA) is expressed as a percentage of the areaat risk (AR). While L-CEFC produces its maximal effect in preventingMI/R injury when used at 0.7 mg/kg, D-CEFC produces the same effect inpreventing MI/R injury when used at 14-fold lower concentration, i.e.,0.05 mg/kg, and produces its maximal effect at 0.1 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

As specified in the Background Section, 3-NT in body fluids and tissuesis a biomarker of the involvement of NO in acute and chronic disorderssuch as I/R injury, atherosclerosis, diabetes, septic shock, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, pulmonary fibrosis,amyotrophic lateral sclerosis (ALS), inflammatory bowel disease,arthritis, allograft rejection, autoimmune myocarditis, pulmonarygranulomatous inflammation, and cancer. Reviewed in, e.g.,Ischiropoulos, Arch. Biochem. Biophys., 1998, 356(1): 1-11; Turko andMurad, Pharmacol. Reviews, 2002, 54(4): 619-634; Radi, Proc. Natl. Acad.Sci. USA, 2004, 101(12): 4003-4008.

The present invention provides isolated short peptides comprising theamino acid sequence Cys-Glu-Phe-His (CEFH; SEQ ID NO: 1) as well asanalogs and derivatives thereof, which peptides efficiently denitratecellular proteins and thus prevent tissue damage associated with excessnitric oxide (NO) and its reactive species. The peptides of theinvention are characterized by a unique combination of stacking, ionicand hydrophobic interactions that allow them to efficiently transfer theNO₂ group from the protein tyrosine to its thiol group and in this wayoffer an unprecedented level of protection against I/R injury and otherconditions associated with excess NO and its reactive species.

In a separate embodiment, the present invention provides a method fortreating I/R injury and other conditions associated with excess NO andits reactive species by exposing nitrated proteins to excess of smallthiols. Examples of small thiols useful in the methods of the inventioninclude, without limitation, homocysteine, N-acetylcysteine [NAC],lipoic acid, thioredoxin [TRX], and Bucillamine. The present inventionalso provides combination treatments using peptides of the invention andsmall thiols.

The short peptides, small thiols, and compositions of the invention haveutility over a wide range of therapeutic applications, and may be usedto treat various types of tissue damage associated with NO and itsreactive species. More specifically, the compounds and compositions ofthis invention may be used to treat disorders including but not limitedto tissue damage associated with I/R injury of various tissues (e.g.,I/R injury of heart muscle associated with heart attack or cardiacsurgery, I/R injury of brain tissue associated with stroke, I/R injuryof liver tissue, skeletal muscles, etc.), septic shock, anaphylacticshock, neurodegenerative diseases (e.g., Alzheimer's and Parkinson'sdiseases), neuronal injury, atherosclerosis, diabetes, multiplesclerosis, autoimmune uveitis, pulmonary fibrosis, oobliterativebronchiolitis, bronchopulmonary dysplasia (BPD), amyotrophic lateralsclerosis (ALS), sepsis, inflammatory bowel disease, arthritis,allograft rejection, autoimmune myocarditis, myocardial inflammation,pulmonary granulomatous inflammation, influenza- or HSV-inducedpneumonia, chronic cerebral vasospasm, allergic encephalomyelitis,central nervous system (CNS) inflammation, Heliobacterium pylongastritis, necrotizing entrerocolitis, celliac disease, peritonitis,early prosthesis failure, inclusion body myositis, preeclampticpregnancies, skin lesions with anaphylactoid purpura, nephrosclerosis,ileitis, leishmaniasis, cancer, and related disorders

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biology,cell biology and protein chemistry within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., “Molecular Cloning: A Laboratory Manual” (2nd Edition, 1989);“DNA Cloning: A Practical Approach, vol. I & II” (D. Glover, ed.);“Oligonucleotide Synthesis” (N. Gait, ed., 1984); “Nucleic AcidHybridization” (B. Hames & S. Higgins, eds., 1985); Perbal, “A PracticalGuide to Molecular Cloning” (1984); Ausubel et al., “Current protocolsin Molecular Biology” (New York, John Wiley and Sons, 1987); andBonifacino et al., “Current Protocols in Cell Biology” (New York, JohnWiley & Sons, 1999).

DEFINITIONS

As used herein, the term “CEFH peptide” is used to refer to any peptideof the invention comprising the sequence Cys-Glu-Phe-His (SEQ ID NO: 1)or any analog or derivative thereof.

As used herein, the term “amino acid” is used to refer to any moleculecontaining an amine and a carboxylic acid. In one embodiment, the aminoacid is attached via a peptide bond.

As used herein in connection with the peptides of the invention, theterms “peptide derivatives” and “peptide analogs” are usedinterchangeably to refer to peptides in which one or more amino acidresidues have been substituted or modified in order (i) to preserve orimprove the unique combination of stacking and/or ionic and/orhydrophobic interactions that allow the CEFH peptides to efficientlydenitrate and/or denitrosylate cellular proteins, and/or (ii) topreserve or improve the delivery of the peptide of the invention to thecells and tissues requiring protein denitration. Peptide derivatives andanalogs according to the present invention include, without limitation,(1) peptides having one or several amino acid replacements with aminoacids or non-natural amino acid analogs having similar properties (suchas, for example, polarity, hydrogen bonding potential, acidic, basic,hydrophobic, aromatic, and the like); (2) peptides produced bycombinatorial shuffling of the relative positions of CEFH amino acids toattain spatial arrangements of the catalysis components that are moreactive than in CEFH peptides; (3) peptides produced by addition of alinker (e.g., homocysteine) instead of cysteine (*CEFH) to provideadditional spatial flexibility and increase the reach of SH group as anacceptor of NO; (4) peptides produced by introduction of electropositivesubstitutions (e.g., CH₃, C₂H₅, tret-Butyl, etc.) into the benzene ringof phenylalanine (CEF*H) to increase the stacking interaction betweenprotein 3-NT and phenylalanine; (5) peptides produced by introduction oftryptophan or cyclic aromatic groups (both natural and synthetic, e.g.,naphtalene, tyrosine, and histidine) instead of phenylalanine(CE[F−>AR*]H) to stabilize the stacking interaction with protein 3-NTbecause of two conjugated aromatic rings; (6) peptides produced bysubstitution (e.g., of phenylalanine) with histidine with or without anelectropositive substitution (e.g., CH₃, C₂H₅, tret-Butyl, etc.)(CE[F−>H]H or (CE[F−>H*]H)) to increase the efficiency of denitration ofthe tyrosine in the protein; (7) peptides produced by modifications thatincrease pKa of glutamic acid (e.g., OH, NO₂, or halide inalpha-position) (CE*FH) to increase the rate of denitration; (8)substitution of glutamic acid with aspartic acid with or withoutmodifications that increase its pKa (e.g., OH, NO₂, or halide inalpha-position) (C[E−>D]FH or C[E−>D*]FH); (9) peptides produced byadditions or substitutions that increase the overall hydrophobicity ofthe peptide and thus render it more cell- and protein globule-permeable.

The term “linker” means any chemical group positioned between the aminoacid segments of the compounds of the present invention. These chemicalgroups may be of any stable chemical structure, and may provide spacingbetween segments, impart conformational constraints on the segments,provide drug targeting or drug delivery functionality, increaseabsorption and/or lifespan in vivo, or provide any additional ancillaryrole that benefits the utility of the molecule. Examples include, butare not limited to, methylene —(CH₂)_(n)— units, polyethylene glycol orother bioploymeric molecules, sugar or carbohydrate moieties, naturalproducts, peptide-nucleic acid (PNA) molecules, both natural andunnatural amino acids and nucleic acids.

Unless otherwise specified, the term “substituted” as used herein refersto substitution with any one or any combination of the followingsubstituents: hydroxy, halogen, carboxyl, cyano, nitro, oxo (═O), thio(═S), substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted aryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedamino, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclylalkyl ring,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted heterocyclic ring, substituted or unsubstituted guanidine,—COOR_(x), —C(O)R_(x), —C(S)R_(x), —C(O)NR_(x)R_(y), —C(O)ONR_(x)R_(y),—NR_(x)CONR_(y)R_(z), —N(R_(x))SOR_(y), —N(R_(x))SO₂R_(y),—(═N—N(R_(x))R_(y)), —NR_(x)C(O)OR_(y), —NR_(x)R_(y), —NR_(x)C(O)R_(y),—NR_(x)C(S)R_(y), —NR_(x)C(S)NR_(y)R_(z), —SONR_(x)R_(y),—SO₂NR_(x)R_(y), —OR_(x), —OR_(x)C(O)NR_(y)R_(z), —OR_(x)C(O)OR_(y),—OC(O)R_(x), —OC(O)NR_(x)R_(y), —R_(x)NR_(y)C(O)R_(z), —R_(x)OR_(y),—R_(x)C(O)OR_(y), —R_(x)C(O)NR_(y)R_(z), —R_(x)C(O)R_(y),—R_(x)OC(O)R_(y), —SR_(x), —SOR_(x), —SO₂R_(x), and —ONO₂, whereinR_(x), R_(y) and R_(z) are independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstituted amino,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted heterocyclylalkyl ring, substituted orunsubstituted heteroarylalkyl, or substituted or unsubstitutedheterocyclic ring. According to one embodiment, the substituents in theaforementioned “substituted” groups cannot be further substituted. Forexample, when the substituent on “substituted alkyl” is “substitutedaryl” the substituent on “substituted aryl” cannot be “substitutedalkenyl”.

The phrases “reactive species of nitric oxide” or “reactive NO species”mean the chemicals capable of nitrosation and nitration of targetmacromolecules, e.g. N₂O₃, N₂O₄, ONOO—, and .NO₂. Peroxynitrite anion(ONOO⁻) and nitrogen dioxide (.NO₂), are formed as secondary products of.NO metabolism in the presence of oxidants including superoxide radicals(O₂.⁻), hydrogen peroxide (H₂O₂), and transition metal centers.

The term “thiol” is used to refer to molecules containing sulfhydryl(—SH) groups that play a role in maintaining the body's redox balanceand defense against oxidants. Examples of small thiols useful in themethods of the present invention include, without limitation, lipoicacid, homocysteine, N-acetylcysteine (NAC), thioredoxin (TRX), andBucillamine.

As used herein, the term “isolated” means that the material beingreferred to has been removed from the environment in which it isnaturally found, and is characterized to a sufficient degree toestablish that it is present in a particular sample. Suchcharacterization can be achieved by any standard technique, such as,e.g., sequencing, hybridization, immunoassay, functional assay,expression, size determination, or the like. Thus, a biological materialcan be “isolated” if it is free of cellular components, i.e., componentsof the cells in which the material is found or produced in nature. Aprotein or peptide that is associated with other proteins and/or nucleicacids with which it is associated in an intact cell, or with cellularmembranes if it is a membrane-associated protein, is considered isolatedif it has otherwise been removed from the environment in which it isnaturally found and is characterized to a sufficient degree to establishthat it is present in a particular sample. A protein or peptideexpressed from a recombinant vector in a host cell, particularly in acell in which the protein is not naturally expressed, is also regardedas isolated.

An isolated organelle, cell, or tissue is one that has been removed fromthe anatomical site (cell, tissue or organism) in which it is found inthe source organism. An isolated material may or may not be “purified”.The term “purified” as used herein refers to a material (e.g., a nucleicacid molecule or a protein) that has been isolated under conditions thatdetectably reduce or eliminate the presence of other contaminatingmaterials. Contaminants may or may not include native materials fromwhich the purified material has been obtained. A purified materialpreferably contains less than about 90%, less than about 75%, less thanabout 50%, less than about 25%, less than about 10%, less than about 5%,or less than about 2% by weight of other components with which it wasoriginally associated.

Methods for purification are well-known in the art. For example,polypeptides can be purified by various methods including, withoutlimitation, preparative disc-gel electrophoresis, isoelectric focusing,HPLC, reverse-phase HPLC, gel filtration, affinity chromatography, ionexchange and partition chromatography, precipitation and salting-outchromatography, extraction, and counter-current distribution. Cells canbe purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting (FACS)). Other purification methods are possible.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to ±20%,preferably up to ±10%, more preferably up to ±5%, and more preferablystill up to ±1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated, the term “about” is implicit and in this context meanswithin an acceptable error range for the particular value.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition. For example, in relation to ischemia/reperfusion (I/R)injury of various tissues, septic shock, anaphylactic shock,neurodegenerative diseases (e.g., Alzheimer's and Parkinson's diseases),neuronal injury, atherosclerosis, diabetes, multiple sclerosis,autoimmune uveitis, pulmonary fibrosis, oobliterative bronchiolitis,bronchopulmonary dysplasia (BPD), amyotrophic lateral sclerosis (ALS),sepsis, inflammatory bowel disease, arthritis, allograft rejection,autoimmune myocarditis, myocardial inflammation, pulmonary granulomatousinflammation, influenza- or HSV-induced pneumonia, chronic cerebralvasospasm, allergic encephalomyelitis, central nervous system (CNS)inflammation, Heliobacterium pylori gastritis, necrotizingentrerocolitis, celliac disease, peritonitis, early prosthesis failure,inclusion body myositis, preeclamptic pregnancies, skin lesions withanaphylactoid purpura, nephrosclerosis, ileitis, leishmaniasis, andcancer treated by the compounds of the present invention, the term“treat” may mean to relieve or alleviate at least one symptom of tissuedamage selected from the group consisting of cellular apoptosis,cellular necrosis, cellular transformation, cellular dysfunction etc.Methods for detecting these symptoms of tissue damage are well known inthe art. For example, as disclosed in the Examples section, below,apoptosis can be detected using an MTS-based assay (using the MTSreagent available from Promega, Madison, Wis.) or the direct counting ofapoptotic cells using flow cytometry. Within the meaning of the presentinvention, the term “treat” also denotes to arrest, delay the onset(i.e., the period prior to clinical manifestation of a disease) and/orreduce the risk of developing or worsening a disease. The term “protect”is used herein to mean prevent, delay or treat, or all, as appropriate,development or continuance or aggravation of a disease in a subject.Within the meaning of the present invention, disease conditions includewithout limitation ischemia/reperfusion (I/R) injury of various tissues(e.g., I/R injury of heart muscle associated with heart attack orcardiac surgery, I/R injury of brain tissue associated with stroke, I/Rinjury of liver tissue, skeletal muscles, etc.), septic shock,anaphylactic shock, neurodegenerative diseases (e.g., Alzheimer's andParkinson's diseases), neuronal injury, atherosclerosis, diabetes,multiple sclerosis, autoimmune uveitis, pulmonary fibrosis,oobliterative bronchiolitis, bronchopulmonary dysplasia (BPD),amyotrophic lateral sclerosis (ALS), sepsis, inflammatory bowel disease,arthritis, allograft rejection, autoimmune myocarditis, myocardialinflammation, pulmonary granulomatous inflammation, influenza- orHSV-induced pneumonia, chronic cerebral vasospasm, allergicencephalomyelitis, central nervous system (CNS) inflammation,Heliobacterium pylori gastritis, necrotizing entrerocolitis, celliacdisease, peritonitis, early prosthesis failure, inclusion body myositis,preeclamptic pregnancies, skin lesions with anaphylactoid purpura,nephrosclerosis, ileitis, leishmaniasis, cancer, and related diseases.

As used herein, the phrase “reduce tissue damage” means reduction of thenecrotic and/or apoptotic area associated with cytotoxic stress, e.g.oxidative stress, inflammation, hypoxia etc. A “significant reduction intissue damage” is a reduction of at least about 10% compared to anappropriate control. As used herein the term “therapeutically effective”applied to dose or amount refers to that quantity of a compound orpharmaceutical composition that is sufficient to result in a desiredactivity upon administration to an animal in need thereof. Within thecontext of the present invention, the term “therapeutically effective”refers to that quantity of a compound or pharmaceutical composition thatis sufficient to reduce or eliminate at least one symptom of tissuedamage selected from the group consisting of cellular apoptosis,cellular necrosis, etc. Methods for detecting these symptoms of tissuedamage are well known in the art. Note that when a combination of activeingredients is administered the effective amount of the combination mayor may not include amounts of each ingredient that would have beeneffective if administered individually.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to ananimal such as a mammal (e.g., a human). Preferably, as used herein, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in mammals, and moreparticularly in humans. “Pharmaceutically acceptable” can also meanconcentrations of the CEFH peptide that do not reduce the concentrationof nitrated/nitrosated proteins below a level required to maintainnormal heart function.

The terms “administering” or “administration” are intended to encompassall means for directly and indirectly delivering a compound to itsintended site of action. The compounds of the present invention can beadministered locally to the affected site (e.g., by direct injectioninto the affected tissue) or systemically. The term “systemic” as usedherein includes parenteral, topical, oral, spray inhalation, rectal,nasal, and buccal administration. Parenteral administration includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,and intracranial administration.

The term “animal” means any animal, including mammals and, inparticular, humans.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise.

COMPOUNDS OF THE INVENTION

The present invention provides isolated short peptides comprising theamino acid sequence Cys-Glu-Phe-His (CEFH; SEQ ID NO: 1) as well asanalogs and derivatives thereof, which peptides efficiently denitratecellular proteins and thus prevent tissue damage associated with excessnitric oxide (NO) and its reactive species. The peptides of theinvention are characterized by a unique combination of stacking, ionicand hydrophobic interactions that allow them to efficiently transfer theNO₂ group from the protein tyrosine to its thiol group and in this wayoffer an unprecedented level of protection against I/R injury and otherconditions associated with excess NO and its reactive species.

The peptides of the present invention are preferably short to allow highaccessibility to various parts of a target protein. More preferably,such peptides are less than eight (8) amino acids long, most preferably,such peptides are four (4) amino acids long. Due to their small size,the peptides of the invention can be easily delivered to essentially alltissues and cells of the body, including cells and tissues separated bythe blood-brain barrier (BBB).

In a specific embodiment, the peptide of the invention has the sequenceCys-Glu-Phe-His (L-CEFH peptide; SEQ ID NO: 1). In another embodiment,the peptide of the invention has the sequence Cys-Glu-Phe-His (SEQ IDNO: 1) and consists of only D-amino acids (D-CEFH peptide). In yetanother embodiment, the peptide of the invention has the sequenceCys-Glu-His-His (CEHH peptide; SEQ ID NO: 2). In further embodiments,the peptide has the sequence Cys-Glu-Phe-His-Cys-Glu-Phe-His (CEFH×2peptide; SEQ ID NO: 3) or contains one or more CEFH peptides fused toone or more CEHH peptides, one or more CEFH peptides fused together, orone or more CEHH peptides fused together. One skilled in the art canenvision additional permutations and combinations of the CEFH and CEHHpeptides that are within the scope of the present invention, includingboth linear and cyclic peptides.

In a second embodiment, the invention provides a compound of formula I,

or a pharmaceutically acceptable salt thereof, wherein the dotted lineis a bond or absent; R¹, R², R³ and R⁴ are independently —C(O)NH—,-(AA)_(n)- and —C(O)-(LL)_(n)-NH—; when the dotted line is a bond,R^(4a) and R^(4b) together are —C(O)NH—, -(AA)_(n)- or—C(O)-(LL)_(n)-NH—; when the dotted line is absent, R^(4a) and R^(4b)are not the same and are independently selected from —C(O)OH and —NH₂;AA is a natural or unnatural amino acid, LL is a linker selected from—(CH₂)_(m)—, —(CH₂CH₂O)_(m)—, —(CH₂CH₂S)_(m)— and —(CH₂CH₂NH)_(m)—, B¹,B², B³ and B⁴ are not the same and are independently selected from

R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from H, C₁-C₆ alkyl,halogen, hydroxy, cyano, nitro, nitoso, amino, sulfhydryl, C₁-C₆ alkoxy,—C(O)O—C₁-C₆ alkyl, —C(O)O—C₁-C₆ aryl, substituted or unsubstitutedaryl, substituted or unsubstituted 5 to 7-membered heterocyclic ring,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted arylcarbonyl, substituted orunsubstituted alkylcarbonyl and substituted or unsubstitutedaminocarbonylalkyl; each occurrence of n is independently an integerfrom 0 to 5; each occurrence of m is independently an integer from 1 to25; and each occurrence of p is independently an integer from 0 to 6.

In another embodiment, B¹, B², B³ and B⁴ are selected from

In a further embodiment, B¹, B², B³ and B⁴ are selected from histidine,phenylalanine, glutamic acid and cysteine.

In one embodiment R¹, R², R³ and R⁴ are all peptide bonds and thecompound is the cyclic tetrapeptide CEFH.

In another embodiment, one of R¹, R², R³ or R⁴ is —NH₂ and —COOHcomprising the N— and C-terminus groups of a linear compound. Forexample, in the case where R¹ is —NH₂ and —COOH, the compound is lineartetrapeptide H₂N—CEFH—COOH. In the case where R² is —NH₂ and —COOH, thecompound is linear tetrapeptide H₂N—HCEF —COOH. In the case where R³ is—NH₂ and —COOH, the compound is linear tetrapeptide H₂N—FHCE-COOH. Inthe case where R⁴ is —NH₂ and —COOH, the compound is linear tetrapeptideH₂N-EFHC—COOH.

In yet another embodiment, R¹, R², R³ or R⁴ are one or more amino acids.For example, if R² is alanine the compound is cyclic-CEFAH. If R² isarginine and R³ is alanine the compound is cyclic-CEAFRH.

In a further embodiment, one or more R¹, R², R³ and R⁴ can be of adifferent chemical nature then a peptide bond or an amino acid. Forexample, the compound may contain an alkyl bridge —(CH₂)_(n)—,polyetheylene glycol —(CH₂CH₂O)_(m)—, —(CH₂CH₂S)_(m)— or—(CH₂CH₂NH)_(m)—, as well as many other derivatives. For example,linkers R¹, R², R³ or R⁴ may be conjugated with additional molecules inorder to achieve a medicinal or pharmacological goal, such as drugtargeting and delivery, increasing in vivo lifespan or improved removalfrom tissues and organs.

In another embodiment, residues R⁵, R⁶, R⁷, R⁸ and R⁹ are modified inone or more available positions with functional group that areindependently selected from H, C₁-C₆ alkyl, halogen, hydroxy, cyano,nitro, nitoso, amino, sulfhydryl, C₁-C₆ alkoxy, —C(O)O—C₁-C₆ alkyl,—C(O)O—C₁-C₆ aryl, substituted or unsubstituted aryl, substituted orunsubstituted 5 to 7-membered heterocyclic ring, substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl,substituted or unsubstituted arylcarbonyl, substituted or unsubstitutedalkylcarbonyl and substituted or unsubstituted aminocarbonylalkyl, whichmay increase denitration activity of the whole molecule.

In a specific embodiment, the amino acid cysteine is replaced withhomo-cysteine, histidine is replaced with arginine, phenylalanine isreplaced with tyrosine, and glutamic acid is replaced with asparticacid. Further, any of these amino acids may be substituted withadditional chemical groups.

In another embodiment, the relative positions of the amino acid residuesmay be altered. For example, compounds such as cyclic-CEFH, cyclic-CEHF,cyclic-CFEH, cyclic-CFHE, cyclic-CHFE and cyclic-CHEF are envisioned.

AA is a natural or unnatural amino acid. In one embodiment, the aminoacid is attached via a peptide bond.

MODIFIED PEPTIDES OF THE INVENTION

The peptides of the invention can be modified in various ways to improvetheir pharmacokinetic and other properties. Peptides can be modified atthe amino (N—) terminus, and/or carboxy (C—) terminus and/or byreplacement of one or more of the naturally occurring geneticallyencoded amino acids with an unconventional amino acid, modification ofthe side chain of one or more amino acid residues, peptidephosphorylation, and the like.

Amino terminus modifications include methylation (e.g., —NHCH₃ or—N(CH₃)₂), acetylation (e.g., with acetic acid or a halogenatedderivative thereof such as α-chloroacetic acid, α-bromoacetic acid, orα-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blockingthe amino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO— or sulfonyl functionality defined by R—SO₂—, where R is selected from alkyl, aryl, heteroaryl, alkyl aryl, andthe like, and similar groups. One can also incorporate a desamino acidat the N-terminus (so that there is no N-terminal amino group) todecrease susceptibility to proteases or to restrict the conformation ofthe peptide compound.

Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. One can also cyclize the peptides ofthe invention, or incorporate a desamino or descarboxy residue at thetermini of the peptide, so that there is no terminal amino or carboxylgroup, to decrease susceptibility to proteases or to restrict theconformation of the peptide. C-terminal functional groups of thecompounds of the present invention include amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lowerester derivatives thereof, and the pharmaceutically acceptable saltsthereof.

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or the stereoisomeric D-amino acids)with other side chains, for instance with groups such as alkyl, loweralkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lowerester derivatives thereof, and with 4-, 5-, 6-, to 7-memberedheterocyclic. For example, proline analogues in which the ring size ofthe proline residue is changed from 5 members to 4, 6, or 7 members canbe employed. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or non-aromatic. Heterocyclic groupspreferably contain one or more nitrogen, oxygen, and/or sulfurheteroatoms. Examples of such groups include the furazanyl, furyl,imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,thiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

Common examples of conventional amino acid replacements includestereoisomers (e.g., D-amino acids) and unnatural amino acids such as,for example, L-ornithine, L-homocysteine, L-homoserine, L-citrulline,3-sulfino-L-alanine, N-(L-arginino)succinate,3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine,triiodothyronine, L-thyroxine, L-selenocysteine, N-(L-arginino)taurine,4-aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a-disubstitutedamino acids, N-alkyl amino acids, lactic acid, β-alanine,3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-methylglycine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,nor-leucine, and other similar amino acids and imino acids. A generalmethod for site-specific incorporation of unnatural amino acids intoproteins and peptides is described in Noren et al., Science, 244:182-188(April 1989).

One can also readily modify peptides by phosphorylation, and othermethods (e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262).

The peptide compounds of the invention also serve as structural modelsfor non-peptidic compounds with similar biological activity. Those ofskill in the art recognize that a variety of techniques are availablefor constructing compounds with the same or similar desired biologicalactivity as the lead peptide compound, but with more favorable activitythan the lead with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis (see, e.g., Morgan and Gainor (1989) Ann.Rep. Med. Chem. 24:243-252). These techniques include replacing thepeptide backbone with a backbone composed of phosphonates, amidates,carbamates, sulfonamides, secondary amines, and N-methylamino acids.

The present invention also provides conjugates of the disclosed peptidemonomers. Thus, according to a preferred embodiment, the monomericpeptides of the present invention are dimerized or oligomerized, therebyenhancing their biological activity.

In one embodiment, the peptide monomers of the invention may beoligomerized using the biotin/streptavidin system. Biotinylated analogsof peptide monomers may be synthesized by standard techniques. Forexample, the peptide monomers may be C-terminally biotinylated. Thesebiotinylated monomers are then oligomerized by incubation withstreptavidin [e.g., at a 4:1 molar ratio at room temperature inphosphate buffered saline (PBS) or HEPES-buffered RPMI medium(Invitrogen) for 1 hour]. In a variation of this embodiment,biotinylated peptide monomers may be oligomerized by incubation with anyone of a number of commercially available anti-biotin antibodies [e.g.,goat anti-biotin IgG from Kirkegaard & Perry Laboratories, Inc.(Washington, D.C.)].

Linkers. In other embodiments, the peptide monomers of the invention canbe dimerized by covalent attachment to at least one linker moiety. Thelinker (L_(K)) moiety can be a C₁₋₁₂ linking moiety optionallyterminated with one or two —NH— linkages and optionally substituted atone or more available carbon atoms with a lower alkyl substituent (e.g.,—NH—R—NH— wherein R is a lower (C₁₋₆) alkylene substituted with afunctional group such as a carboxyl group or an amino group, such as,for example, a lysine residue or a lysine amide).

In an additional embodiment, polyethylene glycol (PEG) may serve as thelinker L_(K) that dimerizes two peptide monomers: for example, a singlePEG moiety may be simultaneously attached to the N-termini of bothpeptide chains of a peptide dimer.

In yet another additional embodiment, the linker (L_(K)) moiety ispreferably, but not necessarily, a molecule containing two carboxylicacids and optionally substituted at one or more available atoms with anadditional functional group such as an amine capable of being bound toone or more PEG molecules. Such a molecule can be depicted as:

—CO—(CH₂)_(n)—X—(CH₂)_(m)—CO—

where n is an integer from 0 to 10, m is an integer from 1 to 10, X isselected from O, S, N(CH₂)_(p)NR₁, NCO(CH₂)_(p)NR₁, and CHNR₁, R₁ isselected from H, Boc, Cbz, etc., and p is an integer from 1 to 10.

Linkers can be incorporated into the peptide during peptide synthesis.For example, where a linker L_(K) moiety contains two functional groupscapable of serving as initiation sites for peptide synthesis and a thirdfunctional group (e.g., a carboxyl group or an amino group) that enablesbinding to another molecular moiety, the linker may be conjugated to asolid support. Thereafter, two peptide monomers may be synthesizeddirectly onto the two reactive nitrogen groups of the linker L_(K)moiety in a variation of the solid phase synthesis technique.

In alternate embodiments where a peptide dimer is dimerized by a linkerL_(K) moiety, said linker may be conjugated to the two peptide monomersof a peptide dimer after peptide synthesis. Such conjugation may beachieved by methods well established in the art. In one embodiment, thelinker contains at least two functional groups suitable for attachmentto the target functional groups of the synthesized peptide monomers. Forexample, a linker with two free amine groups may be reacted with theC-terminal carboxyl groups of each of two peptide monomers. In anotherexample, linkers containing two carboxyl groups, either preactivated orin the presence of a suitable coupling reagent, may be reacted with theN-terminal or side chain amine groups, or C-terminal lysine amides, ofeach of two peptide monomers.

Disulfide Bonds. Generally, although not necessarily, peptide dimerswill also contain one or more intramolecular disulfide bonds betweencysteine residues of the peptide monomers. Preferably, the two monomerscontain at least one intramolecular disulfide bond. Most preferably,both monomers of a peptide dimer contain an intramolecular disulfidebond, such that each monomer contains a cyclic group. Such disulfidebonds may be formed by oxidation of the cysteine residues of the peptidecore sequence. In one embodiment the control of cysteine bond formationis exercised by choosing an oxidizing agent of the type andconcentration effective to optimize formation of the desired isomer. Forexample, oxidation of a peptide dimer to form two intramoleculardisulfide bonds (one on each peptide chain) is preferentially achieved(over formation of intermolecular disulfide bonds) when the oxidizingagent is DMSO. The formation of cysteine bonds can be controlled by theselective use of thiol-protecting groups during peptide synthesis.

Other embodiments of this invention provide for analogues of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isotere for sulfur. These analogues can be preparedfrom the compounds of the present invention, wherein each core sequencecontains at least one C or homocysteine residue and an α-amino-γ-butyricacid in place of the second C residue, via an intramolecular orintermolecular displacement, using methods known in the art (see, e.g.,Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et al. (1991)J. Org. Chem. 56:3146-3149). One of skill in the art will readilyappreciate that this displacement can also occur using other homologs ofα-amino-γ-butyric acid and homocysteine.

In addition to the foregoing cyclization strategies, other non-disulfidepeptide cyclization strategies can be employed. Such alternativecyclization strategies include, for example, amide-cyclizationstrategies as well as those involving the formation of thio-ether bonds.Thus, the compounds of the present invention can exist in a cyclizedform with either an intramolecular amide bond or an intramolecularthio-ether bond. For example, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine and the secondcysteine is replaced with glutamic acid. Thereafter a cyclic monomer maybe formed through an amide bond between the side chains of these tworesidues. Alternatively, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine. A cyclic monomermay then be formed through a thio-ether linkage between the side chainsof the lysine residue and the second cysteine residue of the coresequence. As such, in addition to disulfide cyclization strategies,amide-cyclization strategies and thio-ether cyclization strategies canboth be readily used to cyclize the compounds of the present invention.Alternatively, the amino-terminus of the peptide can be capped with anα-substituted acetic acid, wherein the α-substituent is a leaving group,such as an α-haloacetic acid, for example, α-chloroacetic acid,α-bromoacetic acid, or α-iodoacetic acid.

Spacers. A peptide monomer or dimer may further comprise one or morespacer moieties. Such spacer moieties may be attached to a peptidemonomer or to a peptide dimer (e.g., such spacer moieties may beattached to the linker L_(K) moiety that connects the monomers of apeptide dimer). For example, such spacer moieties may be attached to apeptide via the carbonyl carbon of a lysine linker, or via the nitrogenatom of an iminodiacetic acid linker. Such a spacer may connect apeptide to an attached water soluble polymer moiety or a protectinggroup.

In one embodiment, the spacer moiety is a C₁₋₁₂ linking moietyoptionally terminated with —NH— linkages or carboxyl (—COOH) groups, andoptionally substituted at one or more available carbon atoms with alower alkyl substituent. In one embodiment, the spacer is R—COOH whereinR is a lower (C₁₋₆) alkylene optionally substituted with a functionalgroup such as a carboxyl group or an amino group that enables binding toanother molecular moiety. For example, the spacer may be a glycine (G)residue, or an amino hexanoic acid.

In other embodiments, the spacer is —NH—R—NH— wherein R is a lower(C₁₋₆) alkylene substituted with a functional group such as a carboxylgroup or an amino group that enables binding to another molecularmoiety. For example, the spacer may be a lysine (K) residue or a lysineamide (K—NH₂, a lysine residue wherein the carboxyl group has beenconverted to an amide moiety —CONH₂).

A spacer can be incorporated into the peptide during peptide synthesis.For example, where a spacer contains a free amino group and a secondfunctional group (e.g., a carboxyl group or an amino group) that enablesbinding to another molecular moiety, the spacer may be conjugated to thesolid support. Thereafter, the peptide may be synthesized directly ontothe spacer's free amino group by standard solid phase techniques.

For example, a spacer containing two functional groups is first coupledto the solid support via a first functional group. Next a linker L_(K)moiety having two functional groups capable of serving as initiationsites for peptide synthesis and a third functional group (e.g., acarboxyl group or an amino group) that enables binding to anothermolecular moiety is conjugated to the spacer via the spacer's secondfunctional group and the linker's third functional group. Thereafter,two peptide monomers may be synthesized directly onto the two reactivenitrogen groups of the linker L_(K) moiety in a variation of the solidphase synthesis technique. For example, a solid support coupled spacerwith a free amine group may be reacted with a lysine linker via thelinker's free carboxyl group.

In alternate embodiments where the peptide compounds contain a spacermoiety, said spacer may be conjugated to the peptide after peptidesynthesis. Such conjugation may be achieved by methods well establishedin the art. In one embodiment, the linker contains at least onefunctional group suitable for attachment to the target functional groupof the synthesized peptide. For example, a spacer with a free aminegroup may be reacted with a peptide's C-terminal carboxyl group. Inanother example, a linker with a free carboxyl group may be reacted withthe free amine group of a peptide's N-terminus or of a lysine residue.In yet another example, a spacer containing a free sulfhydryl group maybe conjugated to a cysteine residue of a peptide by oxidation to form adisulfide bond.

Water soluble polymer moieties. The peptide monomers, dimers, ormultimers of the invention may further comprise one or more watersoluble polymer moieties. Preferably, these polymers are covalentlyattached to the peptide compounds of the invention. Included with thebelow description, the U.S. patent application Ser. No. 10/844,933 andInternational Patent Application No. PCT/US04/14887, filed May 12, 2004,are incorporated by reference herein in their entirety.

In recent years, water-soluble polymers, such as polyethylene glycol(PEG), have been used for the covalent modification of peptides oftherapeutic and diagnostic importance. Attachment of such polymers isthought to enhance biological activity, prolong blood circulation time,reduce immunogenicity, increase aqueous solubility, and enhanceresistance to protease digestion (see, e.g., J. M. Harris, Ed.,“Biomedical and Biotechnical Applications of Polyethylene GlycolChemistry,” Plenum, N.Y., 1992; Knauf, et al. (1988) J. Biol. Chem.263;15064; Tsutsumi, et al. (1995) J. Controlled Release 33:447; Kita,et al. (1990) Drug Des. Delivery 6:157; Abuchowski, et al. (1977) J.Biol. Chem. 252:582; Beauchamp, et al. (1983) Anal. Biochem. 131:25;Chen, et al. (1981) Biochim. Biophy. Acta 660:293).

The water soluble polymers useful for the peptide compounds of theinvention may be, for example, polyethylene glycol (PEG), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide copolymers, and polyoxyethylatedpolyols.

The water soluble polymer may be of any molecular weight, and may bebranched or unbranched. A preferred PEG for use in the present inventioncomprises linear, unbranched PEG having a low molecular weight. It isunderstood that in a given preparation of PEG, the molecular weightswill typically vary among individual molecules. Some molecules willweight more, and some less, than the stated molecular weight. Suchvariation is generally reflect by use of the word “about” to describemolecular weights of the PEG molecules.

Peptides, peptide dimers and other peptide-based molecules of theinvention can be attached to water-soluble polymers (e.g., PEG) usingany of a variety of chemistries to link the water-soluble polymer(s) tothe receptor-binding portion of the molecule (e.g., peptide+spacer). Atypical embodiment employs a single attachment junction for covalentattachment of the water soluble polymer(s) to the receptor-bindingportion, however in alternative embodiments multiple attachmentjunctions may be used, including further variations wherein differentspecies of water-soluble polymer are attached to the receptor-bindingportion at distinct attachment junctions, which may include covalentattachment junction(s) to the spacer and/or to one or both peptidechains. In some embodiments, the dimer or higher order multimer willcomprise distinct species of peptide chain (i.e., a heterodimer or otherheteromultimer). By way of example and not limitation, a dimer maycomprise a first peptide chain having a PEG attachment junction and thesecond peptide chain may either lack a PEG attachment junction orutilize a different linkage chemistry than the first peptide chain andin some variations the spacer may contain or lack a PEG attachmentjunction and said spacer, if PEGylated, may utilize a linkage chemistrydifferent than that of the first and/or second peptide chains. Analternative embodiment employs a PEG attached to the spacer portion ofthe receptor-binding portion and a different water-soluble polymer(e.g., a carbohydrate) conjugated to a side chain of one of the aminoacids of the peptide portion of the molecule.

A wide variety of polyethylene glycol (PEG) species may be used forPEGylation of the receptor-binding portion (peptides+spacer).Substantially any suitable reactive PEG reagent can be used. Inpreferred embodiments, the reactive PEG reagent will result in formationof a carbamate or amide bond upon conjugation to the receptor-bindingportion. Suitable reactive PEG species include, but are not limited to,those which are available for sale in the Drug Delivery Systems catalog(2003) of NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu4-chome, Shibuya-ku, Tokyo 150-6019) and the Molecular Engineeringcatalog (2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville,Ala. 35806). For example and not limitation, the following PEG reagentsare often preferred in various embodiments: mPEG2-NHS, mPEG2-ALD,multi-Arm PEG, mPEG(MAL)2, mPEG2(MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA,mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET,heterofunctional PEGs (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS,NHS—PEG-VS, NHS—PEG-MAL), PEG acrylates (ACRL-PEG-NHS),PEG-phospholipids (e.g., mPEG-DSPE), multiarmed PEGs of the SUNBRITEseries including the GL series of glycerine-based PEGs activated by achemistry chosen by those skilled in the art, any of the SUNBRITEactivated PEGs (including but not limited to carboxyl-PEGs, p-NP—PEGs,Tresyl-PEGs, aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs,maleimido-PEGs, hydroxyl-PEG-amine, amino-PEG-COOH,hydroxyl-PEG-aldehyde, carboxylic anhydride type-PEG, functionalizedPEG-phospholipid, and other similar and/or suitable reactive PEGs asselected by those skilled in the art for their particular applicationand usage.

The number of polymer molecules attached may vary; for example, one,two, three, or more water soluble polymers may be attached to a peptideof the invention. The multiple attached polymers may be the same ordifferent chemical moieties (e.g., PEGs of different molecular weight).In some cases, the degree of polymer attachment (the number of polymermoieties attached to a peptide and/or the total number of peptides towhich a polymer is attached) may be influenced by the proportion ofpolymer molecules versus peptide molecules in an attachment reaction, aswell as by the total concentration of each in the reaction mixture. Ingeneral, the optimum polymer versus peptide ratio (in terms of reactionefficiency to provide for no excess unreacted peptides and/or polymermoieties) will be determined by factors such as the desired degree ofpolymer attachment (e.g., mono, di-, tri-, etc.), the molecular weightof the polymer selected, whether the polymer is branched or unbranched,and the reaction conditions for a particular attachment method.

There are a number of PEG attachment methods available to those skilledin the art (see, e.g., Goodson, et al. (1990) Bio/Technology 8:343; EP 0401 384; Malik, et al., (1992) Exp. Hematol. 20:1028-1035; PCT Pub. No.WO 90/12874; U.S. Pat. No. 5,757,078; and U.S. Pat. No. 6,077,939). Forexample, activated PEG may be covalently bound to amino acid residuesvia a reactive group, such as a free amino group in N-terminal aminoacid residues and lysine (K) residues or a free carboxyl group inC-terminal amino acid residues. Sulfhydryl groups (e.g., as found oncysteine residues) may also be used as a reactive group for attachingPEG. In addition, enzyme-assisted methods for introducing activatedgroups (e.g., hydrazide, aldehyde, and aromatic-amino groups)specifically at the C-terminus of a polypeptide have been described(Schwarz, et al. (1990) Methods Enzymol. 184:160; Rose, et al. (1991)Bioconjugate Chem. 2:154; Gaertner, et al. (1994) J. Biol. Chem.269:7224).

For example, PEG molecules may be attached to peptide amino groups usingmethoxylated PEG (“mPEG”) having different reactive moieties. Suchpolymers include mPEG-succinimidyl succinate, mPEG-succinimidylcarbonate, mPEG-imidate, mPEG-4-nitrophenyl carbonate, and mPEG-cyanuricchloride. Similarly, PEG molecules may be attached to peptide carboxylgroups using methoxylated PEG with a free amine group (mPEG-NH₂).

Where attachment of the PEG is non-specific and a peptide containing aspecific PEG attachment is desired, the desired PEGylated compound maybe purified from the mixture of PEGylated compounds. For example, if anN-terminally PEGylated peptide is desired, the N-terminally PEGylatedform may be purified from a population of randomly PEGylated peptides(i.e., separating this moiety from other monoPEGylated moieties).

Site-specific PEGylation at the N-terminus, side chain, and C-terminuscan be performed through (i) solid-phase synthesis (see, e.g., Felix, etal. (1995) Int. J. Peptide Protein Res. 46:253) or (ii) attaching apeptide to extremities of liposomal surface-grafted PEG chains in asite-specific manner through a reactive aldehyde group at the N-terminusgenerated by sodium periodate oxidation of N-terminal threonine (see,e.g., Zalipsky, et al. (1995) Bioconj. Chem. 6:705; this method islimited to polypeptides with N-terminal serine or threonine residues),or (iii) via a hydrazone, reduced hydrazone, oxime, or reduced oximebond is described in U.S. Pat. No. 6,077,939.

In one method, selective N-terminal PEGylation may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, a carbonyl group containing PEG is selectiveattached to the N-terminus of a peptide. For example, one mayselectively N-terminally PEGylate the protein by performing the reactionat a pH which exploits the pK_(a) differences between the ε-amino groupsof a lysine residue and the α-amino group of the N-terminal residue ofthe peptide. By such selective attachment, PEGylation takes placepredominantly at the N-terminus of the protein, with no significantmodification of other reactive groups (e.g., lysine side chain aminogroups). Using reductive alkylation, the PEG should have a singlereactive aldehyde for coupling to the protein (e.g., PEGproprionaldehyde may be used).

Site-specific mutagenesis is a further approach which may be used toprepare peptides for site-specific polymer attachment. By this method,the amino acid sequence of a peptide is designed to incorporate anappropriate reactive group at the desired position within the peptide.For example, WO 90/12874 describes the site-directed PEGylation ofproteins modified by the insertion of cysteine residues or thesubstitution of other residues for cysteine residues.

Where PEG is attached to a spacer or linker moiety, similar attachmentmethods may be used. In this case, the linker or spacer contains areactive group and an activated PEG molecule containing the appropriatecomplementary reactive group is used to effect covalent attachment. Inpreferred embodiments the linker or spacer reactive group contains aterminal amino group (i.e., positioned at the terminus of the linker orspacer) which is reacted with a suitably activated PEG molecule to makea stable covalent bond such as an amide or a carbamate. Suitableactivated PEG species include, but are not limited to,mPEG-para-nitrophenylcarbonate (mPEG-NPC), mPEG-succinimidyl carbonate(mPEG-SC), and mPEG-succinimidyl propionate (mPEG-SPA). In otherpreferred embodiments, the linker or spacer reactive group contains acarboxyl group capable of being activated to form a covalent bond withan amine-containing PEG molecule under suitable reaction conditions.Suitable PEG molecules include mPEG-NH₂ and suitable reaction conditionsinclude carbodiimide-mediated amide formation or the like.

PREPARATION OF THE PEPTIDES OF THE INVENTION

The peptides of the invention may be prepared by classical methods knownin the art. These standard methods include exclusive solid phasesynthesis, automated solid phase synthesis, partial solid phasesynthesis methods, fragment condensation, classical solution synthesis,and recombinant DNA technology (See, e.g., Merrifield J. Am. Chem. Soc.1963 85:2149 and Merrifield et al., 1982, Biochemistry, 21:502).

A preferred method for peptide synthesis is solid phase synthesis. Solidphase peptide synthesis procedures are well-known in the art (see, e.g.,Stewart, Solid Phase Peptide Syntheses, Freeman and Co.: San Francisco,1969; 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA;Goodman, Synthesis of Peptides and Peptidomimetics, Houben-Weyl,Stuttgart 2002). In solid phase synthesis, synthesis is typicallycommenced from the C-terminal end of the peptide using an α-aminoprotected resin. A suitable starting material can be prepared, forinstance, by attaching the required α-amino acid to a chloromethylatedresin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamineresin, or the like. One such chloromethylated resin is sold under thetrade name BIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, Calif.).The preparation of the hydroxymethyl resin has been described(Bodonszky, et al. (1966) Chem. Ind. London 38:1597). Thebenzhydrylamine (BHA) resin has been described (Pietta and Marshall,1970, Chem. Commun., 650), and the hydrochloride form is commerciallyavailable from Beckman Instruments, Inc. (Palo Alto, Calif.). Forexample, an α-amino protected amino acid may be coupled to achloromethylated resin with the aid of a cesium bicarbonate catalyst,according to the method described by Gisin (1973, Helv. Chim. Acta56:1467).

After initial coupling, the α-amino protecting group is removed, forexample, using trifluoroacetic acid (TFA) or hydrochloric acid (HCl)solutions in organic solvents at room temperature. Thereafter, α-aminoprotected amino acids are successively coupled to a growingsupport-bound peptide chain. The α-amino protecting groups are thoseknown to be useful in the art of stepwise synthesis of peptides,including: acyl-type protecting groups (e.g., formyl, trifluoroacetyl,acetyl), aromatic urethane-type protecting groups [e.g.,benzyloxycarboyl (Cbz) and substituted Cbz], aliphatic urethaneprotecting groups [e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl,triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc), allyloxycarbonyl(Alloc), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).

The side chain protecting groups (typically ethers, esters, trityl, PMC(2,2,5,7,8-pentamethyl-chroman-6-sulphonyl), and the like) remain intactduring coupling and is not split off during the deprotection of theamino-terminus protecting group or during coupling. The side chainprotecting group must be removable upon the completion of the synthesisof the final peptide and under reaction conditions that will not alterthe target peptide. The side chain protecting groups for Tyr includetetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and2,5-dichlorobenzyl. The side chain protecting groups for Asp includebenzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl. The sidechain protecting groups for Thr and Ser include acetyl, benzoyl, trityl,tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz. The side chainprotecting groups for Arg include nitro, Tosyl (Tos), Cbz,adamantyloxycarbonyl mesitoylsulfonyl (Mts),2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),4-mthoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side chainprotecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl(2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc.

After removal of the α-amino protecting group, the remaining protectedamino acids are coupled stepwise in the desired order. Each protectedamino acid is generally reacted in about a 3-fold excess using anappropriate carboxyl group activator such as2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate(HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, inmethylene chloride (CH₂Cl₂), N-methyl pyrrolidone, dimethyl formamide(DMF), or mixtures thereof.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagent,such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which notonly cleaves the peptide from the resin, but also cleaves all remainingside chain protecting groups. When a chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides. In preparing the esters of the invention, the resins usedto prepare the peptide acids are employed, and the side chain protectedpeptide is cleaved with base and the appropriate alcohol (e.g.,methanol). Side chain protecting groups are then removed in the usualfashion by treatment with hydrogen fluoride to obtain the desired ester.The resultant peptide can be further purified using HPLC.

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of any of thecompounds of the invention. Synthetic amino acids that can besubstituted into the peptides of the present invention include, but arenot limited to, N-methyl, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl,δ amino acids such as L.-δ-hydroxylysyl and D.-δ-methylalanyl,L-δ-methylalanyl, β amino acids, and isoquinolyl. D-amino acids andnon-naturally occurring synthetic amino acids can also be incorporatedinto the peptides of the present invention.

In addition to chemical synthesis, the peptides of the present inventionmay be synthesized by employing recombinant DNA technology by expressingone or more polynucleotide comprising a peptide coding region. Thus,provided herein are isolated polynucleotides that encode the peptides ofthe present invention as well as recombinant vectors and host cells(both eukaryotic and prokaryotic) that have been genetically modified toexpress or overexpress the peptides of the present invention.

In one embodiment, the invention provides isolated polynucleotidescomprising nucleotide sequences encoding the CEFH peptide of SEQ IDNO: 1. In another embodiment, the invention provides isolatedpolynucleotides comprising nucleotide sequences encoding the CEHHpeptide of SEQ ID NO: 2.

Expression may be achieved in any conventional expression system knownin the art by isolating a DNA fragment encoding the peptide of interestand cloning into an expression vector.

OTHER COMPOUNDS OF THE INVENTION

Useful compounds of the present invention are not limited to peptidesincorporating natural and/or non-natural amino acids. A number ofnon-peptide molecules having similar functional properties can bedeveloped to incorporate disparate chemical functional groups within asingle molecule. These molecules are often referred to as scaffoldingmolecules, or scaffolds, since they can accommodate a wide range ofchemical functionality and can be designed to present the chemicalfunctional groups in a wide array of relative geometric orientations inspace. Molecular scaffold systems include, but are not limited to,carbohydrates (see, e.g., Tamaruya et al., Angew Chem. Int. Ed. Engl.,2004, 43(21):2834-7), peptide nucleic acids (PNA's), (see, e.g., PeptideNucleic Acids: Protocols and Applications, 2nd ed., Peter E. Nielsen,ed., Horizon Bioscience, 2004) and molecules not derived from biologicalprecursors (see, e.g., Savinov and Austin, Org. Lett., 2002,4(9):1419-22).

Chemical functionality comprising these molecules as well as peptides ofthe invention would include (i) at least one thiol group (for example,cysteine or homocysteine), (ii) at least one polar group (for example, afunctional group with a measurable dipole moment, including, but notlimited to, carbonyl groups such as in ketones, esters, or amides, iminegroups alone or in heterocycles, cyano groups, guanidine groups, amidinegroups, etc. as in serine, threonine, lysine, arginine, histidine,tyrosine, tryptophan, glutamic acid, aspartic acid, glutamine orasparagine, cysteine or methionine), (iii) at least one proton donor(such as an alcohol, carboxylic acid, hydroxyl amine, heterocyclic orheteroaromatic NH or OH as in serine, threonine, lysine, arginine,histidine, tyrosine, tryptophan, glutamic acid, aspartic acid, glutamineor asparagine), and (iv) at least one aromatic group (for example,carbocyclic or heteroaromatic groups as in tyrosine, tryptophan,histidine or phenylalanine). It is envisioned that these chemical groupsmay be combined into a single functional group (for example tyrosine,tryptophan, histidine, glutamic acid, aspartic acid, glutamine,asparagine, arginine, and lysine) or be comprised in different portionsof the molecule.

The incorporation of this diverse a set of chemistries may requirechemical protection of reactive functionality during synthesis. Thesetechniques are well known in the art and can be found in references suchas T. W. Green, P. G. M. Wuts, Protective Groups in Organic Synthesis,Wiley-Interscience, New York, 1999.

COMPOSITION COMPRISING ONE OR MORE COMPOUND(S) OF THE INVENTION

Tyrosine denitrating compounds disclosed herein may be formulated ascompositions together with a pharmaceutically acceptable carrier (suchas an adjuvant or vehicle) and/or excipient, and/or diluent Acomposition of this invention may include pharmaceutically acceptablesalts of the components therein. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe peptide) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic and the like. Salts formed with the free carboxylgroups can be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like.

Pharmaceutically acceptable carriers are familiar to those skilled inthe art and can include sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution saline solutions and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. For compositionsformulated as liquid solutions, acceptable carriers and diluents includesaline and sterile water, and may optionally include antioxidants,buffers, bacteriostats, and other common additives. The compositions canalso be formulated as pills, capsules, granules, or tablets which maycontain, in addition to a peptide of this invention, diluents,dispersing and surface active agents, binders, and lubricants. Properformulation is dependent upon the route of administration chosen.

METHODS FOR INDENTIFYING ADDITONAL PEPTIDES SUITABLE FOR THE TREATMENTOF DISEASES ASSOCIATED WITH THE ACCUMULATION OF 3-NITROTYROSINE

The present invention also provides methods for generating and/oridentifying novel peptides having the same or better functionalcharacteristics as the CEFH peptides of the invention using thefollowing design methods (aimed at preserving or improving the uniquecombination of stacking and/or ionic and/or hydrophobic interactionsthat allow the CEFH peptides to efficiently transfer the NO₂ group fromthe protein tyrosine to its thiol group):

-   (1) Combinatorial shuffling of the relative positions of CEFH amino    acids to attain spatial arrangements of the catalysis components    that are more active.-   (2) Addition of a linker (e.g., homocysteine) instead of cysteine    (*CEFH) to provide additional spatial flexibility and increase the    reach of SH group as an acceptor of NO. This modification may also    increase the overall hydrophobicity of the peptide and thus render    it more cell- and protein globule-permeable.-   (3) Introduction of electropositive substitutions (e.g., CH₃, C₂H₅,    tret-Butyl, etc.) into the benzene ring of phenylalanine (CEF*H) to    increase the stacking interaction between protein 3-NT and    phenylalanine. This modification also increases the overall    hydrophobicity of the peptide and thus renders it more cell- and    protein globule-permeable.-   (4) Introduction of tryptophan or cyclic aromatic groups (both    natural and synthetic, e.g., naphtalene, tyrosine, and histidine)    instead of phenylalanine (CE[F−>AR*]H) to stabilize the stacking    interaction with protein 3-NT because of two conjugated aromatic    rings.-   (5) Substitution of phenylalanine with one or more histidines    (CE[F−>H]H) with or without an electropositive substitution (e.g.,    CH₃, C₂H₅, tret-Butyl, etc.) to increase the efficiency of    denitration of the tyrosine of the protein;-   (6) Modification that increases pKa of glutamic acid (e.g., OH, NO₂,    or halide in alpha-position) (CE*FH) to increase of the rate of    denitration.-   (7) Substitution of glutamic acid with aspartic acid with or without    modifications that increase its pKa (e.g., OH, NO₂, or halide in    alpha-position) (C[E−>D]FH or C[E−>D*]FH);

The present invention further provides in vitro and in vivo methods forfunctional testing of the novel denitrating peptides generated using theabove methods, comprising:

-   (1) in vitro testing by adding the peptide to a nitrated protein    having 3-NT (e.g., ONOO-nitrated albumin) and monitoring the    disappearance of 3-NT (e.g., by immunoblotting using anti-3-NT    antibodies);-   (2) testing by adding the peptide to a cell culture treated with NO    and/or reactive NO species (e.g., subject to NO/ONOO-exposure) and    measuring cell survival by methods such as an MTS-based assay (using    the MTS reagent available from Promega) or the direct counting of    apoptotic cells using flow cytometry.-   (3) testing by adding the peptide to a cell culture treated with NO    and/or reactive NO species (e.g., subject to NO/ONOO-exposure) and    measuring the disappearance of 3-NT;-   (4) in vivo testing by administering the peptide to an animal model    of a relevant disease (e.g., I/R injury, septic shock, Alzheimer's    disease, etc.) and monitoring disease progression;-   (5) in vivo testing by administering the peptide to an animal model    for I/R injury and determining the size of the infarct using a    p-nitro-blue tetrazolium (NBT)-based assay while also monitoring    functional parameters such as heart rate, mean arterial blood    pressure, cardiac output, etc. Identification of improved peptides    of the invention can be conducted in a format of high-throughput    screening (HTS) assays, including both cell-based and cell-free    assays. See, e.g., Furka et al. (14th International Congress of    Biochemistry, 1988, Volume #5, Abstract FR:013; Furka, Int. J.    Peptide Protein Res. 1991; 37:487-493), U.S. Pat. Nos. 4,631,211 and    5,010,175.

METHODS FOR THE USE OF THE COMPOUNDS OF THE INVENTION AND COMPOSITIONSTHEREOF

The compounds of the invention and compositions thereof are useful in awide variety of therapeutic applications including, but not limited to,the treatment of tissue damage associated with NO and its reactivespecies. For example, the compounds and compositions of the presentinvention can be used to treat diseases including, but not limited to,tissue damage associated with I/R injury of various tissues (e.g., I/Rinjury of heart muscle associated with heart attack or cardiac surgery,I/R injury of brain tissue associated with stroke, I/R injury of livertissue, skeletal muscles, etc.), septic shock, anaphylactic shock,neurodegenerative diseases (e.g., Alzheimer's and Parkinson's diseases),neuronal injury, atherosclerosis, diabetes, multiple sclerosis,autoimmune uveitis, pulmonary fibrosis, oobliterative bronchiolitis,bronchopulmonary dysplasia (BPD), amyotrophic lateral sclerosis (ALS),sepsis, inflammatory bowel disease, arthritis, allograft rejection,autoimmune myocarditis, myocardial inflammation, pulmonary granulomatousinflammation, influenza- or HSV-induced pneumonia, chronic cerebralvasospasm, allergic encephalomyelitis, central nervous system (CNS)inflammation, Heliobacterium pylori gastritis, necrotizingentrerocolitis, celliac disease, peritonitis, early prosthesis failure,inclusion body myositis, preeclamptic pregnancies, skin lesions withanaphylactoid purpura, nephrosclerosis, ileitis, leishmaniasis, cancer,and related disorders.

Such methods include administering a composition of this invention to ananimal/patient in an amount effective to treat tissue damage.

The optimal therapeutically effective amount of a compound orcomposition of this invention may be determined experimentally, takinginto consideration the exact mode of administration, the form in whichthe drug is administered, the indication toward which the administrationis directed, the subject involved (e.g., body weight, health, age, sex,etc.), and the preference and experience of the physician orveterinarian in charge.

As disclosed herein, the concentrations of CEFH peptides administered inthe present invention are both therapeutically effective andpharmaceutically acceptable. The L-CEFH peptide of the present inventionis preferably used to treat or prevent tissue damage in vivo at 0.1-3.5mg/kg, most preferably at 0.7 mg/kg. The D-CEFH peptide of the presentinvention is preferably used at 0.01-0.5 mg/kg, most preferably at 0.1mg/kg.

The efficacy of the peptides and compositions of this invention can bedetermined using the in vitro and in vivo assays described in theExamples section, below.

Following methodologies which are well-established in the art, effectivedoses and toxicity of the peptides and compositions of the presentinvention, which performed well in in vitro tests, can be determined instudies using small animal models (e.g., mice, rats or dogs) in whichthey have been found to be therapeutically effective and in which thesedrugs can be administered by the same route proposed for the humantrials.

For any pharmaceutical composition used in the methods of the invention,dose-response curves derived from animal systems can be used todetermine testing doses for administration to humans. In safetydeterminations for each composition, the dose and frequency ofadministration should meet or exceed those anticipated for use in anyclinical trial.

As disclosed herein, the dose of the compound in the compositions of thepresent invention is determined to ensure that the dose administeredcontinuously or intermittently will not exceed an amount determinedafter consideration of the results in test animals and the individualconditions of a patient. A specific dose naturally varies (and isultimately decided according to the judgment of the practitioner andeach patient's circumstances) depending on the dosage procedure, theconditions of a patient or a subject animal such as age, body weight,sex, sensitivity, feed, dosage period, drugs used in combination,seriousness of the disease, etc.

Toxicity and therapeutic efficacy of the compositions of the inventioncan be determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between therapeutic and toxic effects isthe therapeutic index and it can be expressed as the ratio ED₅₀/LD₅₀.

DELIVERY OF THE PEPTIDES OF THE INVENTION TO THE TARGET DAMAGED TISSUE

All known peptide delivery methods can be used to deliver the peptidesof the present invention to the target damaged cells and tissues. Thespecific type of delivery useful for a given peptide is determined byits specific size, flexibility, conformation, biochemical properties ofconstituent amino acids, and amino acid arrangement. Peptide compositionalso determines, in part, the degree of protein binding, enzymaticstability, cellular sequestration, uptake into non-target tissue,clearance rate, and affinity for protein carriers. Other aspectsindependent of peptide composition must also be considered, such ascerebral blood flow, diet, age, sex, species (for experimental studies),dosing route, and effects of existing pathological conditions.

Examples of delivery methods useful for obtaining effective tissuedelivery of the peptides of the invention (and effective passage throughthe blood-brain-barrier [BBB] in case of brain tissues), include,without limitation (reviewed, e.g., in Witt and Davis, AAPS Journal,2006; 8(1): E76-E88.):

-   (i) invasive procedures (e.g., direct injection [e.g., using an    external pump or i.v. line], transient osmotic opening, shunts, and    biodegradable implants);-   (ii) pharmacologically-based approaches to increase the tissue    delivery by chemical modification of the peptide molecule itself, or    by the attachment or encapsulation of the peptide in a substance    that increases permeability, stability, bioavailability, and/or    receptor affinity; in addition, modification of a peptide structure    and/or addition of constituents (e.g., lipophilicity enhancers,    polymers, antibodies) may enhance local peptide concentration in the    target tissue;-   (iii) physiologic-based strategies which exploit various carrier    mechanisms; these strategies can be combined, dependent of the    nature of a given peptide, creating “hybrid” peptides, resulting in    synergistic delivery and end-effect.

Specific examples of peptide modifications and methods useful forimproving delivery of the peptides of the invention include, withoutlimitation, lipidization (e.g., methylation, dimethylation, orhalogenation of constituent amino acids or acylation or alkylation ofthe N-terminal amino acid), structural modification to enhance stability(e.g., use of D-amino acids, N-acylation, or cyclization, e.g., via adisulfide-bridge or via a hydrazide bridge), glycosylation (e.g., addingsimple sugars such as, e.g., glucose or xylose), increasing affinity fornutrient transporters (e.g., adding hexose or large neutral amino acidcarriers which facilitate delivery of substrates to the brain), forminga prodrug by conjugating a peptide to a molecule with a knowntransporter activity or to a lipophilicity enhancer, which is cleaved ator near the site of action (e.g., using esterification [with, e.g.,aromatic benzoyl esters or branched chain tertiary butyl esters] oramidation of amino, hydroxyl, or carboxylic acid-containing peptides;also, redox system-mediated delivery to the brain may be facilitatedusing conjugation to a methyldihydropyridine carrier and subsequentoxidation by NADH-linked dehydrogenases in the brain, which results in aquaternary ammonium salt, which does not cross back through the BBBendothelium), vector-based delivery (e.g., by coupling a peptide to asubstance that increases the affinity to and transport across biologicalmembranes via receptor-mediated or absorptive-mediated endocytosisfollowed by peptide release via enzymatic cleavage [e.g., conjugation ofa peptide to murine monoclonal antibody (OX26) to the transferrin orconjugation to cationized albumin to increase brain uptake]),cationization to increase membrane entry via absorptive-mediatedendocytosis, and polymer conjugation/encapsulation (e.g., conjugation topoly(ethylene glycol) [PEG] or poly(styrene maleic acid) orencapsulation via micro- or nano-particles [e.g., polymericnanoparticles ranging in size between 10 and 1000 nm, which have apolysorbate overcoating such as, e.g., polysorbate-80], liposomes [e.g.,surface-modified long-circulating liposomes grafted with a flexiblehydrophilic polymer such as, e.g., PEG and/or liposomes composed of aphospholipid bilayer such as, e.g., pluronic copolymer P85, that act asa carrier for both hydrophilic and hydrophobic peptides], micelles[e.g., stable polymeric micelles prepared from amphiphilicPEG-phospholipid conjugates], or cell ghosts). Reviewed in Torchilin andLukyanov, DDT, 2003, 8(6): 259-266; Egleton and Davis, NeuroRx, 2005, 2:44-53; Witt and Davis, AAPS Journal, 2006; 8(1): E76-E88.

Regardless of the delivery method used, an important aspect of thepresent invention is to keep the size of the resulting delivered peptidesufficiently small (e.g., by using cleavable conjugates) to facilitateits access to various regions within the nitrated target protein.

Oral Delivery. Contemplated for use herein are oral solid dosage forms,which are described generally in Remington's Pharmaceutical Sciences,18th Ed. 1990 (Mack Publishing Co. Easton, Pa. 18042) at Chapter 89,which is herein incorporated by reference. Solid dosage forms includetablets, capsules, pills, troches or lozenges, cachets, pellets,powders, or granules. Also, liposomal or proteinoid encapsulation may beused to formulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979, herein incorporated by reference. In general, the formulationwill include a peptide of the invention (or chemically modified formsthereof) and inert ingredients which allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

Also contemplated for use herein are liquid dosage forms for oraladministration, including pharmaceutically acceptable emulsions,solutions, suspensions, and syrups, which may contain other componentsincluding inert diluents; adjuvants such as wetting agents, emulsifyingand suspending agents; and sweetening, flavoring, and perfuming agents.

As discussed above, the peptides may be chemically modified so that oraldelivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe component molecule itself, where said moiety permits (a) increase inpeptide stability (e.g., by inhibition of proteolysis) and (b) efficientuptake into the blood stream from the stomach or intestine. As discussedabove, common delivery-improving peptide modifications includePEGylation or the addition of moieties such as propylene glycol,copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane (see, e.g.,Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymesas Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.4:185-189).

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunum, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. Preferably, the release willavoid the deleterious effects of the stomach environment, either byprotection of the peptide (or derivative) or by release of the peptide(or derivative) beyond the stomach environment, such as in theintestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic (i.e. powder), for liquid forms a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide (or derivative) can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs, or even as tablets.These therapeutics could be prepared by compression.

Colorants and/or flavoring agents may also be included. For example, thepeptide (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the peptide (or derivative)with an inert material. These diluents could include carbohydrates,especially mannitol, lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress, and Avicel.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. The disintegrants may also be insoluble cationicexchange resins. Powdered gums may be used as disintegrants and asbinders. and can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the peptide (or derivative) agent togetherto form a hard tablet and include materials from natural products suchas acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC)could both be used in alcoholic solutions to granulate the peptide (orderivative).

An antifrictional agent may be included in the formulation of thepeptide (or derivative) to prevent sticking during the formulationprocess. Lubricants may be used as a layer between the peptide (orderivative) and the die wall, and these can include but are not limitedto; stearic acid including its magnesium and calcium salts,polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils andwaxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the peptide (or derivative) into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrosefatty acid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the peptide (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Controlled release oral formulations may be desirable. The peptide (orderivative) could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation.Some enteric coatings also have a delayed release effect. Another formof a controlled release is by a method based on the Oros therapeuticsystem (Alza Corp.), i.e. the drug is enclosed in a semipermeablemembrane which allows water to enter and push drug out through a singlesmall opening due to osmotic effects.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The peptide (orderivative) could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Parenteral delivery. Preparations according to this invention forparenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, or emulsions. Examples of non-aqueous solventsor vehicles are propylene glycol, polyethylene glycol, vegetable oils,such as olive oil and corn oil, gelatin, and injectable organic esterssuch as ethyl oleate. Such dosage forms may also contain adjuvants suchas preserving, wetting, emulsifying, and dispersing agents. They may besterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, immediately before use.

Preferably, the L-peptides of the invention (e.g., L-CEFH) areadministered to treat diseases related to NO damage by parental i.v.injection in a standard physiological solution. The D-peptides of theinvention (e.g., D-CEFH) can be administered using any standardadministration technique known in the art, such as oral administration.

The peptides can also be delivered using a vector (such as a viralvector) with the ability to express a peptide of this invention.

Combination Therapy

The novel anti-tissue damage peptides and compositions of the presentinvention can be used in conjunction with small thiols (such as, e.g.,homocysteine, N-acetylcysteine (NAC), lipoic acid, thioredoxin (TRX),Bucillamine, etc., where a preferred small thiol is α-lipoic acid) aswell as existing anti-inflammatory therapeutics such as inhibitors ofTNF-α, inhibitors of COX-1/COX-2, inhibitors of IL-1β, etc. In aspecific embodiment, the novel therapeutics of the invention areadministered in combination with Non-Steroidal Anti-Inflammatory Drugs(NSAIDs). Suitable NSAIDs include, but are not limited to, those whichinhibit cyclooxygenase, the enzyme responsible for the biosyntheses ofthe prostaglandins and certain autocoid inhibitors, including inhibitorsof the various isoenzymes of cyclooxygenase (including, but not limitedto, cyclooxygenase-1 and -2), and as inhibitors of both cyclooxygenaseand lipoxygenase relates to NSAID, such as the commercially availableNSAIDs aceclofenac, acemetacin, acetaminophen, acetaminosalol,acetyl-salicylic acid, acetyl-salicylic-2-amino-4-picoline-acid,5-aminoacetylsalicylic acid, alclofenac, aminoprofen, amfenac, ampyrone,ampiroxicam, anileridine, bendazac, benoxaprofen, bermoprofen,α-bisabolol, bromfenac, 5-bromosalicylic acid acetate, bromosaligenin,bucloxic acid, butibufen, carprofen, celecoxib, chromoglycate,cinmetacin, clindanac, clopirac, sodium diclofenac, diflunisal, ditazol,droxicam, enfenamic acid, etodolac, etofenamate, felbinac, fenbufen,fenclozic acid, fendosal, fenoprofen, fentiazac, fepradinol, flufenac,flufenamic acid, flunixin, flunoxaprofen, flurbiprofen, glutametacin,glycol salicylate, ibufenac, ibuprofen, ibuproxam, indomethacin,indoprofen, isofezolac, isoxepac, isoxicam, ketoprofen, ketorolac,lornoxicam, loxoprofen, meclofenamic acid, mefenamic acid, meloxicam,mesalamine, metiazinic acid, mofezolac, montelukast, nabumetone,naproxen, niflumic acid, nimesulide, olsalazine, oxaceprol, oxaprozin,oxyphenbutazone, paracetamol, parsalmide, perisoxal,phenyl-acethyl-salicylate, phenylbutazone, phenylsalicylate, pyrazolac,piroxicam, pirprofen, pranoprofen, protizinic acid, reserveratol,salacetamide, salicylamide, salicylamide-O-acetyl acid, salicylsulphuricacid, salicin, salicylamide, salsalate, sulindac, suprofen,suxibutazone, tamoxifen, tenoxicam, tiaprofenic acid, tiaramide,ticlopridine, tinoridine, tolfenamic acid, tolmetin, tropesin, xenbucin,ximoprofen, zaltoprofen, zomepirac, tomoxiprol, zafirlukast andcyclosporine. Additional NSAID genera and particular NSAID compounds aredisclosed in U.S. Pat. No. 6,297,260 and International PatentApplication No. WO 01/87890.

EXAMPLES

The present invention will be better understood by reference to thefollowing non-limiting examples.

Example 1 Identification of CEFH Peptide as Efficient Compound forMediating Denitration of 3-Nitrotyrosine In Vitro

The CEFH peptides were designed using the free docking software program,ArgusLab 4.0 (available on the World Wide Web atplanaria-software.com/arguslab40.htm), to calculate the interactionenergy between molecules. L-CEFH peptide and D-CEFH peptide weresynthesized and purified by New England Peptides Inc (Gardner, Mass.)and GL Biochem Ltd. (Shanghai), respectively, using a standard solidphase Fmoc protocol. D-CEFH was synthesized since this peptide is lessprone to proteolysis compared to its L-CEFH counterpart. Peptideswithout His or Cys did not work.

Several groups have previously reported an in vivo denitrationphenomenon (e.g., Kamisaki et al (1998) Proc Natl Acad Sci USA 95:11584-11589; Aulak et al (2004) Am J Physiol Heart Circ Physiol 286:H30-H38). However, they tried to isolate denitration activity using pure3-NT and failed. The present inventors have hypothesized that thestability of pure 3-NT is much higher than the stability of 3-NTincorporated in a protein and used ONOO-nitrated albumin as thesubstrate for detecting denitration activity.

To test the ability of L-CEFH peptide (Cys-Glu-Phe-His; SEQ ID NO: 1) tomediate 3-NT denitration in vitro, 20 μM CEFH peptide solutioncontaining 10 mM Tris HCl pH 7.2 was added to albumin previouslynitrated by peroxynitrite (ONOO⁻). Denitration of the peroxynitrite(ONOO)-nitrated albumin following 0, 3, or 10 minutes of incubation withL-CEFH peptide was then visualized by dot-immunoblotting with antibodiesagainst 3-NT. As shown in FIG. 2, essentially complete denitration(i.e., >90%) was achieved after 10 minutes of treatment with 20 μML-CEFC peptide solution containing 10 mM Tris HCl pH 7.2 at atemperature of 25° C.

Example 2 CEFH Peptide Efficiently Reduces Cell Death Mediated by NitricOxide and Peroxynitrite

The ability of the L-CEFH peptide (Cys-Glu-Phe-His; SEQ ID NO: 1) toreduce cell death caused by nitric oxide (NO) and peroxynitrite (ONOO⁻)was tested in cell culture. Following treatment with NO/ONOO⁻, HeLacells were incubated with either a control buffer, 5 mM cysteine, or 20μM L-CEFH peptide for 40 minutes. The percentage of cells surviving wasdetermined by a MTS-based assay (available from Promega, Madison, Wis.).As shown in FIG. 3, as compared to “no treatment” control, both cysteine(Cys, at 5 mM) and L-CEFH peptide (4P, at 20 μM) improved HeLa cellsurvival to the same extent (i.e., by 60-80%). Importantly, the L-CEFHpeptide showed the same efficacy as cysteine, when used at 250 timeslower concentration.

Example 3 CEFH Peptide Reduces Myocardial Ischemia-Reperfusion Injuryand 3-Nitrotyrosine Accumulation In Vivo

The effect of the L-CEFH peptide (Cys-Glu-Phe-His; SEQ ID NO: 1) on theextent of myocardial ischemia-reperfusion (MI/R) injury and on theaccumulation of 3-nitrotyrosine (3-NT) in myocardial tissue uponmyocardial infarction was tested in a rat model in vivo.

Myocardial ischemia (MI) was produced in anaesthetized adult male Wistarrats (300-350 grams) under controlled ventilation. After thoracotomy atthe fourth intercostal space, the heart was exteriorized and a 6-0 silkligature was passed under the left coronary artery with a needle. After30 minutes of MI, the ligature was released and the myocardium wasreperfused for 1 hour. Rats were randomized 5 minutes after thebeginning of reperfusion to receive either vehicle (control, n=6) orL-CEFH peptide (0.7 mg/kg; n=9) i.v. The extent of MI/R injury, orinfarct size, was determined by calculating the ratio of the area ofnecrosis (NA) to the area at risk (AR). Accumulation of 3-NT in thetested heart tissue corresponding to the area at risk was analyzed byWestern dot-immunoblotting of total protein (extracted from the area atrisk) with antibodies against 3-NT.

As shown in FIG. 4, the L-CEFH peptide (4P) efficiently prevents MI/Rinjury in a rat model of myocardial infarction. The decrease in theinfarct size in the presence of the L-CEFH peptide is 3-5 fold ascompared to the control.

As shown in FIG. 5, the L-CEFH peptide prevents accumulation of 3-NT inthe heart tissue in a rat model of myocardial infarction. As compared tothe control, in the peptide-treated I/R animals, the 3-NT accumulationis reduced by about 70%.

Example 4 D-CEFH Peptide has a Superior Activity in Reducing MyocardialIschemia-Reperfusion Injury as Compared to L-CEFH

To determine whether CEFH activity can be improved when replacingL-amino acids with D-amino acids, D-CEFH peptide consisting of allD-amino acids (Cys-Glu-Phe-His; SEQ ID NO: 1) was generated and itsactivity was compared to the activity of the original L-CEFH peptidecontaining all L-amino acids in vivo in a rat model of myocardialinfarction.

Rat model of myocardial infarction was generated as disclosed in Example3, above. 0, 0.02, 0.05, 0.1, 0.14, 0.35, and 0.7 mg/kg of D-CEFHpeptide (n≦4 for each concentration) were administered i.v. The extentof MI/R injury, or infarct size, was determined by calculating the ratioof the area of necrosis (NA) to the area at risk (AR).

As shown in FIG. 6, D-CEFH peptide has a superior activity in preventingMI/R injury as compared to L-CEFH peptide in a rat model of MI. WhileL-CEFC produces its maximal effect in preventing MI/R injury when usedat 0.7 mg/kg, the D-CEFH peptide produces the same effect in preventingMI/R injury when used at 14-fold lower concentration, i.e., 0.05 mg/kg,and produces its maximal effect at 0.1 mg/kg.

Example 5 Investigation of Long-Term Protective Effects of CEFH Peptideon Standard Heart Functional Parameters in a Rat Model of MyocardialInfarction

Rat model of myocardial infarction is generated as disclosed in Example3, above. ECG is performed to monitor the standard heart functionalparameters 1, 2, 3, 4 weeks after I/R surgery.

Example 6 Protective Effects of CEFH Peptide in a Mouse Model ofAnaphylactic Shock

Anaphylactic shock is a sudden, life-threatening allergic reactionassociated with severe hypotension. Excessive production of thevasodilator NO contributes to this inflammatory hypotension and shock.

Mouse models of anaphylactic shock are created generally as outlined inCauwels et al., J. Clin. Invest., 2006, 116(8): 2244-2251. FemaleC57BL/6 mice are housed in temperature-controlled, air-conditionedfacilities with 14-hour light/10-hour dark cycles and food and water adlibitum. All data are collected using mice 8-12 weeks of age.

PAF-induced anaphylactic shock model. Platelet-activating factor (PAF)is implicated in the cardiovascular dysfunctions occurring in variousshock syndromes, including anaphylaxis. Intravenous PAF injection inconscious mice elicits rapid shock and results in death within 20-30minutes. In the model, mice are injected intravenously with 55 μg PAF(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; Sigma-Aldrich).Mortality is scored up to 7 days after challenge. In experimentalanimals, 0.1 mg/kg D-CEFH peptide (Cys-Glu-Phe-His; SEQ ID NO: 1) isadministered i.v. 1 hour before PAF.

BSA/OVA-induced anaphylactic shock model. Mice are first sensitized withBSA or OVA in the presence of adjuvants. Two different sensitizationmodels are used:

-   (a) Mice are given a single i.p. injection of 1 mg BSA    (Sigma-Aldrich) mixed with 300 ng pertussis toxin (Sigma-Aldrich).    Anaphylaxis is elicited 15 days later by i.v. injection of 2 mg of    BSA.-   (b) Mice are sensitized by i.p. injection of 100 μg OVA    (Sigma-Aldrich), aluminum hydroxide (Sigma-Aldrich, 1 mg) and    pertussis toxin (300 ng). Mice are challenged 19-20 days later by    i.v. injection of 150 μg OVA.

Soon after challenge, mice develop severe hypothermia and rapidlysuccumb to systemic shock reaction. Mortality is scored up to 7 daysafter challenge. In experimental animals, the D-CEFH peptide isadministered by i.v. injection 2 hours prior to challenge with a lethaldose of BSA or OVA.

1. A compound of formula I

or a pharmaceutically acceptable salt thereof, wherein the dotted lineis a bond or absent; R¹, R², and R³ are independently —C(O)NH—,-(AA)_(n)- or —C(O)-(LL)_(n)-NH—; when the dotted line is a bond, R^(4a)and R^(4b) together are —C(O)NH—, -(AA)_(n)- or —C(O)-(LL)_(n)-NH—; whenthe dotted line is absent, R^(4a) and R^(4b) are not the same and areindependently selected from —C(O)OH and —NH₂; AA is a natural orunnatural amino acid; LL is a linker selected from —(CH₂)_(m)—,—(CH₂CH₂O)_(m)—, —(CH₂CH₂S)_(m)— and —(CH₂CH₂NH)_(m)—; B¹, B², B³ and B⁴are not the same and are independently selected from

R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from H, C₁-C₆ alkyl,halogen, hydroxy, cyano, nitro, nitoso, amino, sulfhydryl, C₁-C₆ alkoxy,—C(O)O—C₁-C₆ alkyl, —C(O)O—C₁-C₆ aryl, substituted or unsubstitutedaryl, substituted or unsubstituted 5 to 7-membered heterocyclic ring,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted arylcarbonyl, substituted orunsubstituted alkylcarbonyl and substituted or unsubstitutedaminocarbonylalkyl; each occurrence of n is independently an integerfrom 0 to 5; each occurrence of m is independently an integer from 1 to25; and each occurrence of p is independently an integer from 0 to
 6. 2.The compound according to claim 1, wherein B¹, B², B³ and B⁴ areselected from


3. The compound according to claim 1, wherein B¹, B², B³ and B⁴ areselected from histidine, phenylalanine, glutamic acid and cysteine. 4.An isolated peptide comprising the amino acid sequence Cys-Glu-Phe-His(SEQ ID NO: 1) or analogs or derivatives thereof, wherein said peptideis between four amino acids and eight amino acids long.
 5. The peptideof claim 4 having the amino acid sequence Cys-Glu-Phe-His (SEQ ID NO: 1)or analogs or derivatives thereof.
 6. The peptide of claim 4 having theamino acid sequence Cys-Glu-Phe-His (SEQ ID NO: 1).
 7. The peptide ofclaim 5, wherein all amino acids are D-amino acids.
 8. The peptide ofclaim 4, wherein at least one amino acid is D-amino acid.
 9. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 10. A method for stimulatingdenitration of 3-nitrotyrosine in a cell or tissue of an animal, saidmethod comprising administering to the cell or tissue the compound ofclaim
 1. 11. A method for preventing cell death induced by nitric oxideand its reactive species, said method comprising administering to thecell the compound of claim
 1. 12. A method for preventingischemia-reperfusion injury in a tissue of an animal, said methodcomprising administering to the tissue the compound of claim
 1. 13. Amethod for treating a disorder manifested by accumulation of3-nitrotyrosine in an animal, said method comprising administering tothe animal a therapeutically effective amount of the compound ofclaim
 1. 14. The method of claim 13, wherein the disorder is selectedfrom the group consisting of ischemia/reperfusion (I/R) injury,atherosclerosis, diabetes, septic shock, Alzheimer's disease,Parkinson's disease, anaphylactic shock, neuronal injury,atherosclerosis, diabetes, multiple sclerosis, autoimmune uveitis,pulmonary fibrosis, oobliterative bronchiolitis, bronchopulmonarydysplasia (BPD), amyotrophic lateral sclerosis (ALS), sepsis,inflammatory bowel disease, arthritis, allograft rejection, autoimmunemyocarditis, myocardial inflammation, pulmonary granulomatousinflammation, influenza- or HSV-induced pneumonia, chronic cerebralvasospasm, allergic encephalomyelitis, central nervous system (CNS)inflammation, Heliobacterium pylori gastritis, necrotizingentrerocolitis, celliac disease, peritonitis, early prosthesis failure,inclusion body myositis, preeclamptic pregnancies, skin lesions withanaphylactoid purpura, nephrosclerosis, ileitis, leishmaniasis, andcancer.
 15. The method of claim 13, wherein the disorder isischemia/reperfusion (I/R) injury.
 16. The method of claim 13, whereinthe amount of the compound administered is within the range of 0.01-3.5milligrams per kilogram.
 17. (canceled)
 18. (canceled)
 19. (canceled)20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. Apharmaceutical composition comprising the peptide of claim 4 and apharmaceutically acceptable carrier.
 29. A method for stimulatingdenitration of 3-nitrotyrosine in a cell or tissue of an animal, saidmethod comprising administering to the cell or tissue the peptide ofclaim
 4. 30. A method for preventing cell death induced by nitric oxideand its reactive species, said method comprising administering to thecell the peptide of claim
 4. 31. A method for preventingischemia-reperfusion injury in a tissue of an animal, said methodcomprising administering to the tissue the peptide of claim
 4. 32. Amethod for treating a disorder manifested by accumulation of3-nitrotyrosine in an animal, said method comprising administering tothe animal a therapeutically effective amount of the peptide of claim 4.33. The method of claim 32, wherein the disorder is selected from thegroup consisting of ischemia/reperfusion (I/R) injury, atherosclerosis,diabetes, septic shock, Alzheimer's disease, Parkinson's disease,anaphylactic shock, neuronal injury, atherosclerosis, diabetes, multiplesclerosis, autoimmune uveitis, pulmonary fibrosis, oobliterativebronchiolitis, bronchopulmonary dysplasia (BPD), amyotrophic lateralsclerosis (ALS), sepsis, inflammatory bowel disease, arthritis,allograft rejection, autoimmune myocarditis, myocardial inflammation,pulmonary granulomatous inflammation, influenza- or HSV-inducedpneumonia, chronic cerebral vasospasm, allergic encephalomyelitis,central nervous system (CNS) inflammation, Heliobacterium pylorigastritis, necrotizing entrerocolitis, celliac disease, peritonitis,early prosthesis failure, inclusion body myositis, preeclampticpregnancies, skin lesions with anaphylactoid purpura, nephrosclerosis,ileitis, leishmaniasis, and cancer.
 34. The method of claim 32, whereinthe amount of the compound administered is within the range of 0.01-3.5milligrams per kilogram.